This protocol describes the isolation of keratinocytes from pregnant bald mice. It can also be used for isolation of primary rat keratinocytes.

Suppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. This protocol describes the preparation of a subtracted cDNA or genomic DNA library, and includes methods for cDNA synthesis, tester and driver DNA digestion, and adapter ligation.

Suppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. This protocol describes a method for subtractive hybridization using adapter-ligated tester and RsaI-digested driver DNA samples.

Suppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. In this protocol, differentially presented DNAs are selectively amplified using PCR. It is strongly recommended that subtractions be performed in both directions for each tester/driver DNA pair. Forward subtraction is designed to enrich for differentially presented molecules present in the tester but not in the driver; reverse subtraction is designed to enrich for differentially presented sequences present in the driver but not in the tester. Therefore, each experiment should have at least four reactions: (1) subtracted tester DNAs, (2) unsubtracted tester control, (3) reverse-subtracted tester DNAs, and (4) unsubtracted control for the reverse subtraction.

Suppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. The major drawback of SSH is the presence of background clones that represent nondifferentially expressed DNA species in the subtracted libraries. In some cases, the number of background clones may considerably exceed the number of target clones. This protocol describes mirror orientation selection (MOS)--a simple procedure that substantially decreases the number of background clones in libraries generated by SSH. The MOS technique is based on the rationale that after PCR amplification, during SSH, background molecules will be present in one orientation only, relative to the adapter sequences. Genuine SSH clones will be present in both sequence orientations.

Suppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. This protocol describes a method for the use of PCR-based DNA dot blots in the differential screening of arrayed subtracted DNA clones. For high-throughput screening, a 96-well or 384-well format PCR from one of several thermal cycler manufacturers is recommended. Alternatively, single tubes can be used.

Suppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. This protocol describes a method for screening a subtracted cDNA library by differential hybridization using (1) a tester-specific subtracted probe (forward-subtracted probe), (2) a driver-specific subtracted probe (reverse-subtracted probe), (3) a cDNA probe synthesized directly from tester mRNA (or tester genomic DNA), and (4) a cDNA probe synthesized directly from driver mRNA (or driver genomic DNA).

Flowering in Arabidopsis thaliana is modulated by a number of environmental factors. It is therefore important to perform flowering-time measurements under controlled environmental conditions, including light quality and intensity, day length, temperature, watering, and spacing between plants. Ideally, pots of plants are rotated, and individual plants of different genotypes are placed randomly within the experimental setup. Because of the large influence of environmental effects, it is imperative that only plants grown at the same time and in the same place be compared. Flowering is also modulated by endogenous factors and can thus be affected by mutations in genes that are not primarily involved in the control of flowering time. The variation of flowering time between ecotypes is generally large. Therefore, only those mutants that are induced in the same background can be compared. This protocol describes a method for determining the flowering time of Arabidopsis plants.

The primary goal of the International Haplotype Map Project has been to develop a haplotype map of the human genome that describes the common patterns of genetic variation, in order to accelerate the search for the genetic causes of human disease. Within the project, ~3.9 million distinct single-nucleotide polymorphisms (SNPs) have been genotyped in 270 individuals from four worldwide populations. The project data are available for unrestricted public use at the HapMap website. This site, which is the primary portal to genotype data produced by the project, offers bulk downloads of the data set, as well as interactive data browsing and analysis tools that are not available elsewhere. Research into the genetic contributions to a human disease commonly focuses on candidate genes identified from linkage and/or association studies, as well as from pathways suspected to be involved in a particular disease process. In studying candidate genes, a researcher will want to know whether there are any common SNPs in the immediate vicinity, what those SNPs’ alleles are, and the relative frequencies of the alleles in the population. The researcher will also be particularly interested in coding SNPs, whose alleles change the amino acid sequence of the gene product and therefore might represent functional variations. This protocol provides details on how to use the genome browser to navigate to and explore HapMap data for a gene or region of interest.

The primary goal of the International Haplotype Map Project has been to develop a haplotype map of the human genome that describes the common patterns of genetic variation, in order to accelerate the search for the genetic causes of human disease. Within the project, ~3.9 million distinct single-nucleotide polymorphisms (SNPs) have been genotyped in 270 individuals from four worldwide populations. The project data are available for unrestricted public use at the HapMap website. This site, which is the primary portal to genotype data produced by the project, offers bulk downloads of the data set, as well as interactive data browsing and analysis tools that are not available elsewhere. In many cases, a researcher will be interested in downloading HapMap data from a region of interest for local analysis. This protocol describes the direct download of genotype, frequency, tag-SNPs, and other reports from the project website, using the genome browser.

The primary goal of the International Haplotype Map Project has been to develop a haplotype map of the human genome that describes the common patterns of genetic variation, in order to accelerate the search for the genetic causes of human disease. Within the project, ~3.9 million distinct single-nucleotide polymorphisms (SNPs) have been genotyped in 270 individuals from four worldwide populations. The project data are available for unrestricted public use at the HapMap website. This site, which is the primary portal to genotype data produced by the project, offers bulk downloads of the data set, as well as interactive data browsing and analysis tools that are not available elsewhere. Advanced users who wish to exercise fine control over the display of regions of high linkage disequilibrium (LD) or who wish to experiment with new algorithms for tag-SNP picking may wish to analyze HapMap data using the HaploView program. This program works well in combination with the HapMap website genome browser. A big advantage of HaploView over the genome browser is that it displays simultaneous high- and low-power views of regions of LD, and gives immediate feedback during scrolling and zooming operations. This protocol describes the use of HaploView to manipulate HapMap data.

The primary goal of the International Haplotype Map Project has been to develop a haplotype map of the human genome that describes the common patterns of genetic variation, in order to accelerate the search for the genetic causes of human disease. Within the project, ~3.9 million distinct single-nucleotide polymorphisms (SNPs) have been genotyped in 270 individuals from four worldwide populations. The project data are available for unrestricted public use at the HapMap Web site. This site, which is the primary portal to genotype data produced by the project, offers bulk downloads of the data set, as well as interactive data browsing and analysis tools that are not available elsewhere. Because of performance considerations, interactive access to HapMap data via the genome browser on the HapMap Web site is limited to regions no more than 5 Mb wide. Researchers who wish to obtain data for chromosome- or genome-wide data have two choices: Bulk download or HapMart access. HapMart, described in this protocol, allows researchers to select SNPs using diverse criteria and to display just those aspects of the data set that they are interested in.

The primary goal of the International Haplotype Map Project has been to develop a haplotype map of the human genome that describes the common patterns of genetic variation, in order to accelerate the search for the genetic causes of human disease. Within the project, ~3.9 million distinct single-nucleotide polymorphisms (SNPs) have been genotyped in 270 individuals from four worldwide populations. The project data are available for unrestricted public use at the HapMap Web site. This site, which is the primary portal to genotype data produced by the project, offers bulk downloads of the data set, as well as interactive data browsing and analysis tools that are not available elsewhere. Bulk downloads of chromosome- or genome-wide data provide text dumps of the entire HapMap data set. Although complete, such downloads do not provide any filtering or selection services. This protocol describes the retrieval of HapMap data via bulk download.

Strict regulation of transcription factor activity is essential to establish and maintain gene expression. Eukaryotic cells control transcription factors at many different levels. Post-translational regulatory mechanisms (e.g., phosphorylation, nuclear translocation, multimerization, regulated degradation, etc.) play particularly important roles because they enable cells to respond to various intra- and extracellular stimuli quickly and without prior protein synthesis. However, extensive post-translational changes can make it difficult to identify differentially regulated transcription factors. Common genomic screening techniques such as DNA microarray analysis are unable to detect any mode of regulation beyond that of mRNA stability. This protocol describes the differential display of DNA-binding proteins (DDDP), which is based on the electrophoretic mobility shift assay (EMSA) and detects DNA-binding transcription factors, independent of the number or nature of regulatory steps required for activation. DDDP is an unbiased screening technique that can be used in any experimental system that uses concentrated protein extracts. A plasmid library containing random DNA sequences is constructed. This library is then used to generate radioactive DNA probes to test protein extracts from different sources in parallel for differentially regulated DNA-binding proteins. Plasmids corresponding to probes that display differential DNA-binding activity can be sequenced, and the binding sequence can be narrowed down in a two-step procedure. The corresponding transcription factors can then be identified by bioinformatic and/or biochemical methods.

The GAL4 system is a method for ectopic gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. This protocol describes the generation of driver and reporter lines for use with the GAL4 system in Drosophila. A promoter-GAL4 fusion is constructed using a P-element transformable vector, and a GAL4-responsive target gene is created via generation of an upstream activation sequence (UAS)-reporter construct. An alternative strategy for integration using the phiC31 system is also provided. Transformant lines are generated using standard procedures for microinjection.

Section in situ hybridization (SISH) is a high-resolution tool used to analyze gene expression patterns. This protocol utilizes the Tecan Freedom EVO150 platform to perform high-throughput SISH on paraffin sections to detect mRNA with a digoxigenin (DIG)-labeled probe. The slide is mounted and imaged before performing immunohistochemistry (IHC) on the same section. The dual reaction enables a marker of protein expression to be localized on the same section as the mRNA and facilitates more accurate annotation of the gene expression.

This protocol describes a method to reduce the numbers of fibroblasts from adherent populations of epithelial cells by using differential trypsinization.

This protocol describes a method for performing reduction and S-alkylation of Coomassie-blue-stained proteins within an intact gel. Reduction is performed with dithiothreitol, and alkylation with 4-vinylpyridine. (Treatment of free cysteines with 4-vinylpyridine yields the S-β-(4-pyridylethyl) cysteinyl derivative.) S-β-(4-pyridylethyl) cysteine-containing peptides can be readily identified during RP-HPLC by their characteristic absorbance at 254 nm and during electrospray ionization tandem mass spectrometry by the appearance of a characteristic pyridylethyl fragment ion of 10⁶ Da. The position of cysteine residues in a polypeptide sequence can be determined either by mass spectrometry or as phenylthiohydantoin S-β-(4-pyridylethyl) cysteine during Edman degradation.

This protocol describes a method for performing reduction and S-alkylation of Coomassie-blue-stained proteins within individually excised, stained protein spots or bands. Reduction is performed with dithiothreitol, and alkylation with 4-vinylpyridine. (Treatment of free cysteines with 4-vinylpyridine yields the S-β-(4-pyridylethyl) cysteinyl derivative.) S-β-(4-pyridylethyl) cysteine-containing peptides can be readily identified during RP-HPLC by their characteristic absorbance at 254 nm and during electrospray ionization tandem mass spectrometry by the appearance of a characteristic pyridylethyl fragment ion of 10⁶ Da. The position of cysteine residues in a polypeptide sequence can be determined either by mass spectrometry or as phenylthiohydantoin S-β-(4-pyridylethyl) cysteine during Edman degradation.

The hormone auxin is transported directionally through the plant body to effect a variety of morphological processes. In the embryo, auxin is required in the early stages of development to establish the bilateral axis of the developing embryo. Later, it participates in vascular element patterning and differentiation, lateral organ outgrowth in the root and shoot, and local growth responses to external stimuli such as light and gravity. A convenient way to measure auxin response, described in this protocol, is to monitor the effects of auxins or of auxin transport inhibitors on root growth.

Brassinosteroid (BR) is required for normal plant growth and development; most notably, BR promotes cell elongation. Thus, mutants with defects in BR biosynthesis or response have defects in cell elongation. They fail to undergo skotomorphogenesis (etiolation) in the dark and they are dwarves when grown in the light. Whether a cell elongation defect can be rescued, unaffected, or alleviated by BR treatment provides an important criterion for whether a mutant has a defect in BR biosynthesis or BR response or whether the mutant phenotype is unrelated to BR. On the other hand, mutants with increased BR signaling are less sensitive to the BR-biosynthesis inhibitor brassinazole (BRZ). The hypocotyl length of dark-grown seedlings and petiole length of light-grown plants are often measured to determine BR responsiveness; BRZ inhibition of BR biosynthesis can reduce hypocotyl and petiole elongation. Wild-type seedlings grown on 1 µM BRZ in the dark have hypocotyls approximately one-third the length of those on medium lacking BRZ. This protocol describes the measurement of BR responsiveness, with or without BRZ, in light- and dark-grown seedlings.

The flux of ions across membranes via ion channels is vital to cellular responses to internal and external stimuli, and therefore to cellular survival in changing circumstances. Patch clamping is a powerful technique for ion channel investigation, because it enables measurement of both net ion fluxes across the entire surface area of a cell and ion currents flowing through a single open channel. However, unlike animal cells, plant cells are surrounded by cell walls that prevent the physical contact between the patch pipette and the plasma membrane necessary for the patch clamp technique. To demonstrate how patch clamping can be applied to plant physiology research, we describe a protocol used to record potassium ion (K⁺) channel currents in Arabidopsis guard cell protoplasts (a widely studied model cell type in plant biology). The protocol requires a two-step cellulase and pectinase digestion to isolate high quality Arabidopsis guard cell protoplasts (i.e., plant cells lacking their cell walls), preparation of suitable glass capillary microelectrodes, and formation of the whole-cell configuration with a gigaohm (G) seal. We also describe the history of the protocol and list other types of plant cells from which successful patch clamp recordings have been obtained.

In post-embedding methods of immunogold staining, the cells or tissues are fixed chemically or cryoimmobilized, dehydrated, and embedded in epoxy or acrylic resins. Thin sections (50-70 nm in thickness) are cut using an ultramicrotome with a diamond knife, using a water bath to collect the sections as they slide off the knife. The sections are stretched with solvent vapor or a heat source and collected onto either bare or plastic-coated nickel grids. The sections are then stained immunochemically with primary antibodies raised against antigens exposed on the surface of the sections. The primary antibodies are visualized by staining immunochemically with secondary antibodies raised against the species and isotype of the primary antibodies, conjugated to colloidal gold particles. The immunochemically stained sections are then contrast stained with salts of uranium (uranyl acetate) and lead (lead citrate) to reveal the ultrastructure of the cells, and are finally viewed by transmission electron microscopy (TEM). Chemical fixation and embedding in a highly cross-linked epoxy resin is the method of choice for optimal ultrastructure and stability of the thin section in the electron beam. Immunogold staining of thin epoxy resin sections, described here, is useful if the antigen of interest is very resistant to fixative, or if only archived material that was fixed primarily for ultrastructural studies is available.

In post-embedding methods of immunogold staining, the cells or tissues are fixed chemically or cryoimmobilized, dehydrated, and embedded in epoxy or acrylic resins. Thin sections (50-70 nm in thickness) are cut using an ultramicrotome with a diamond knife, using a water bath to collect the sections as they slide off the knife. The sections are stretched with solvent vapor or a heat source and collected onto either bare or plastic-coated nickel grids. The sections are then stained immunochemically with primary antibodies raised against antigens exposed on the surface of the sections. The primary antibodies are visualized by staining immunochemically with secondary antibodies raised against the species and isotype of the primary antibodies, conjugated to colloidal gold particles. The immunochemically stained sections are then contrast stained with salts of uranium (uranyl acetate) and lead (lead citrate) to reveal the ultrastructure of the cells, and are finally viewed by transmission electron microscopy (TEM). LR White was introduced as a low-toxicity alternative to epoxy resins, which frequently contained carcinogens. Unlike the simplest acrylic resins, in which monomers are polymerized to form long chains, the LR resins contain aromatic cross-linkers to improve the stability of the sections under the electron beam. LR White and Gold both have very low viscosity and readily penetrate, even into dense tissue. In this protocol, aldehyde-fixed tissue is dehydrated in ethanol, impregnated in LR White resin and polymerized under vacuum or in a nitrogen atmosphere before sectioning and immunogold staining.

In this method for freeze substitution and low-temperature embedding in resin prior to immunogold staining, lightly fixed tissue pieces are cryoprotected by immersion in polypropylene glycol. The cryoprotected tissues are quench frozen and transferred under liquid nitrogen to vials containing frozen methanol or methanol containing uranyl acetate. The vials are transferred to a substitution vessel where the temperature can be controlled and a nitrogen atmosphere maintained. The temperature is raised (typically at 5°C/h to -90°C) and maintained for 24 h. This temperature is cold enough to prevent recrystallization of water and thus tissue disruption, but high enough for movement of water to occur and allow substitution with the liquid methanol. After 24 h, ~90% of the water has been substituted. The substitution medium is replaced and the temperature is raised to -70°C for 24 h. The substitution medium is changed again and the temperature is raised to -50°C. The tissue is impregnated with Lowicryl HM20 over a period of 1-5 d and the resin is polymerized by UV irradiation. Tissue is then sectioned and stained immunochemically with primary antibodies raised against antigens exposed on the surface of the sections, and primary antibodies are visualized by staining with secondary antibodies conjugated to colloidal gold particles. The immunochemically stained sections are contrast stained with uranyl acetate and lead citrate to reveal the ultrastructure of the cells, and are finally viewed by transmission electron microscopy (TEM). This is the simplest and most versatile of the post-embedding procedures.

A pre-embedding method of immunochemical staining is used if antigens are damaged by resin embedding, or if the best preservation of membranes is required. Applying immunogold reagents to sections of lightly fixed tissue, free of embedding medium, can be a very sensitive method of immunochemical staining. Cells or tissues are fixed as strongly as possible and then treated with a cryoprotectant, which is usually a mixture of sucrose and polyvinylpyrrolidone (PVP). They are frozen onto pins in liquid nitrogen and sectioned at approximately -100°C. The frozen sections are thaw-mounted on to Formvar/nickel film grids and the cryoprotectant is removed by floating the grids on drops of phosphate-buffered saline (PBS). The immunogold staining is performed on the unembedded sections, which are subsequently contrast counterstained and infiltrated with a mixture of methylcellulose and uranyl acetate. In this protocol, samples are sectioned at low temperature, thaw-mounted onto film grids, immunochemically stained, contrast counterstained, and embedded/encapsulated in situ on the grid before viewing by transmission electron microscopy (TEM).

Fluorescent in situ hybridization (FISH) is commonly used to analyze the three-dimensional distribution of RNAs in intact embryos and tissues. Tyramide signal amplification (TSA) significantly increases the sensitivity and resolution of FISH probe signals. This protocol includes optimized TSA-FISH procedures for Drosophila embryos, ovaries, and larval tissues. Instructions are given for the preparation of RNA probes, the collection and fixation of tissues, and the hybridization and TSA-mediated detection of probes, including options for high-throughput processing in 96-well plates. Variations of the procedure for RNA-RNA and RNA-protein costaining are also described.

The use of the enzyme alkaline phosphatase allows identification of phosphopeptides in a mixture of predominantly nonphosphopeptides. Using a MALDI-MS instrument, the masses of peptides are acquired both before and after alkaline phosphatase treatment, which removes phospho-moieties from serine, threonine, and/or tyrosine. (Any peptide whose mass decreases by 80 Da, or a multiple thereof, is a phosphopeptide.) An advantage of using MALDI-MS for these experiments is that the peptide ions produced tend to be singly charged rather than multiply charged (as with ESI), thus making the interpretation easier. This protocol describes a method for in-solution dephosphorylation prior to MALDI-MS analysis.

The use of the enzyme alkaline phosphatase allows identification of phosphopeptides in a mixture of predominantly nonphosphopeptides. Using a MALDI-MS instrument, the masses of peptides are acquired both before and after alkaline phosphatase treatment, which removes phospho-moieties from serine, threonine, and/or tyrosine. (Any peptide whose mass decreases by 80 Da, or a multiple thereof, is a phosphopeptide.) An advantage of using MALDI-MS for these experiments is that the peptide ions produced tend to be singly charged rather than multiply charged (as with ESI), thus making the interpretation easier. This protocol describes a method for in-solution dephosphorylation after MALDI-MS analysis.

The use of the enzyme alkaline phosphatase allows identification of phosphopeptides in a mixture of predominantly nonphosphopeptides. Using a MALDI-MS instrument, the masses of peptides are acquired both before and after alkaline phosphatase treatment, which removes phospho-moieties from serine, threonine, and/or tyrosine. (Any peptide whose mass decreases by 80 Da, or a multiple thereof, is a phosphopeptide.) An advantage of using MALDI-MS for these experiments is that the peptide ions produced tend to be singly charged rather than multiply charged (as with ESI), thus making the interpretation easier. This protocol describes on-probe dephosphorylation following MALDI-MS analysis.

The majority of mouse chromosome preparations for banding are now made by air-drying and, in essence, require the production of a cell suspension as a starting point. Some samples such as blood cultures, ascitic fluids, or cells growing in suspension will already be in suspension; others, such as bone marrow, solid tumors, or cells growing as attached layers in culture must be converted to suspensions. The basic steps in karyotyping and banding embryonal carcinoma cells are outlined below.

Although the large polytene chromosomes of diptera were originally described in the late 1880s, it was not until the early 1930s that their significance to the study of the genome of Drosophila was realized. Polytene chromosomes are found in several larval and adult tissues, but preparations are usually made of the chromosomes in the larval salivary glands, because the glands are easily dissected and the polytene chromosomes are large.

The formidable size and structure of polytene chromosomes allow mapping of chromosomal protein distributions at very high resolution. This protocol describes the preparation of polytene chromosomes from Drosophila larvae, immunostaining of the chromosomes with a protein of interest, and counterstaining with Giemsa and Hoechst.

Seed germination requires breaking the dormancy that is maintained by abscisic acid (ABA) and involves the activation of gibberellin (GA) biosynthesis and signaling. Mutants with low germination rates may be defective in GA biosynthesis and/or signaling and often require exogenous GAs for efficient germination. Conversely, mutants with either increased GA signaling or reduced signaling by the GA antagonist ABA are resistant to GA-biosynthesis inhibitors or ABA, respectively. Mutants with enhanced GA signaling or decreased ABA content are also more resistant to inhibition of germination by Paclobutrazol (PAC), a GA biosynthesis inhibitor. This protocol describes the measurement of the germination rate of Arabidopsis seeds in the presence of GA, ABA, or PAC.

Hematoxylin and eosin (H&E) stains have been used for at least a century and are still essential for recognizing various tissue types and the morphologic changes that form the basis of contemporary cancer diagnosis. The stain has been unchanged for many years because it works well with a variety of fixatives and displays a broad range of cytoplasmic, nuclear, and extracellular matrix features. Hematoxylin has a deep blue-purple color and stains nucleic acids by a complex, incompletely understood reaction. Eosin is pink and stains proteins nonspecifically. In a typical tissue, nuclei are stained blue, whereas the cytoplasm and extracellular matrix have varying degrees of pink staining. Well-fixed cells show considerable intranuclear detail. Nuclei show varying cell-type- and cancer-type-specific patterns of condensation of heterochromatin (hematoxylin staining) that are diagnostically very important. Nucleoli stain with eosin. If abundant polyribosomes are present, the cytoplasm will have a distinct blue cast. The Golgi zone can be tentatively identified by the absence of staining in a region next to the nucleus. Thus, the stain discloses abundant structural information, with specific functional implications. A limitation of hematoxylin staining is that it is incompatible with immunofluorescence. It is useful, however, to stain one serial paraffin section from a tissue in which immunofluorescence will be performed. Hematoxylin, generally without eosin, is useful as a counterstain for many immunohistochemical or hybridization procedures that use colorimetric substrates (such as alkaline phosphatase or peroxidase). This protocol describes H&E staining of tissue and cell sections.

This protocol describes the sectioning of tissues embedded in paraffin blocks. Paraffin sections require extensive fixation and processing steps but provide superior morphology compared with other sectioning methods. Sectioning paraffin blocks requires experience and should be learned from an experienced researcher, if possible.

It is imperative that the slides and coverslips used in fluorescence microscopy procedures be extremely clean. Although coverslips look clean, especially when a new box is first opened, they may have a thin film of grease on them that will not allow tissue culture cells to adhere well and that may interfere with some processing steps in certain protocols. Therefore, coverslips should routinely be washed with acid or base solutions to rid them of this film. Commercial precleaned slides are also likely to be dirty and must be washed prior to use. This protocol describes various approaches for cleaning slides and coverslips and sterilizing them for cell culture, as well as methods for subbing slides. In the subbing procedure, slides are coated with gelatin, aminoalkylsilane, or poly-L-lysine solution to promote the adhesion of cells or tissues to the glass surface. Gelatin or aminoalkylsilane is usually used for tissue sections or small organisms, whereas poly-L-lysine is routinely used for cultured cells.

This protocol describes a method for embedding tissues in paraffin blocks for sectioning. Paraffin sections require extensive fixation and processing steps, but provide superior morphology compared with other sectioning methods.

Paraffin sections of bone usually require a decalcification step after fixation before sectioning. This protocol describes a method for decalcifying fixed tissue.

T7-based linear amplification of DNA (TLAD) uses a linear amplification approach based on in vitro transcription (IVT) of template DNA by RNA polymerase from T7 phage. TLAD was designed for use with the ChIP-chip method (whereby DNA recovered from chromatin immunoprecipitation [ChIP] of cell lysate is used for subsequent analysis on DNA microarrays) and requires nanogram quantities of dsDNA to generate microgram amounts of amplified RNA. In Part I of the method, described here, a 3' conserved end is added to the template dsDNA, using terminal deoxynucleotidyl transferase (TdT) tailing. The initial treatment with calf intestinal phosphatase (CIP) is optional but strongly recommended for removing 3' phosphate groups, because most genomic DNA fragmentation methods (i.e., sonication, micrococcal nuclease digestion, and certain restriction digests) produce a significant proportion of 3' phosphate groups within the mixture of fragmented genomic DNA. This protocol is compatible with the presence of RNase A and can be carried out immediately after digestion of RNA carried over from ChIP, without any intermediate clean-up step. The tailing reaction involves the addition of a short (20-40 nucleotide [nt]) poly(dT) tail to the template DNA. The included dideoxynucleotide acts as a tail terminator in the reaction mixture and is necessary to maintain a tight size distribution. This poly(dT) tail provides a conserved 3' element that permits the addition of a T7 promoter sequence in the subsequent second-strand synthesis step. IVT can then use this newly appended T7 promoter and linearly amplify the template dsDNA, producing amplified RNA product.

T7-based linear amplification of DNA (TLAD) uses a linear amplification approach based on in vitro transcription (IVT) of template DNA by RNA polymerase from the T7 phage. TLAD was designed primarily for use with the ChIP-chip method (whereby DNA recovered from chromatin immunoprecipitation [ChIP] of cell lysate is used for subsequent analysis on DNA microarrays) and requires nanogram quantities of dsDNA to generate microgram amounts of amplified RNA. Briefly, the strategy is to add a 3' conserved end to the template dsDNA, using terminal deoxynucleotidyl transferase (TdT) tailing, which permits the addition of a T7 promoter sequence in the subsequent second-strand synthesis step, described here. At this stage, the strand-displacement activity of the Klenow fragment polymerase separates the two strands of the template DNA, after which the enzyme performs fill-in 5' -> 3' polymerization. Its 3' -> 5' exonuclease activity may also remove the 3' overhanging poly(dT) tails, although the efficiency of this activity will vary based on the length of the poly(dT) tail. IVT can then use this newly appended T7 promoter. Because the T7-based IVT proceeds as an isothermal reaction, it linearly amplifies the template DNA, producing antisense RNA (aRNA) (i.e., each strand of RNA produced is antisense to the original template strand). Since both strands are amplified, this distinction is usually not important and is affected only by the location of the T7 promoter and poly(A) tract on the aRNA.

T7-based linear amplification of DNA (TLAD) uses a linear amplification approach based on in vitro transcription (IVT) of template DNA by RNA polymerase from the T7 phage. TLAD was designed primarily for use with the ChIP-chip method (whereby DNA recovered from chromatin immunoprecipitation [ChIP] of cell lysate is used for subsequent analysis on DNA microarrays) and requires nanogram quantities of dsDNA to generate microgram amounts of amplified RNA. Briefly, the strategy is to add a 3' conserved end to the template dsDNA, using terminal deoxynucleotidyl transferase (TdT) tailing, which permits the addition of a T7 promoter sequence in the subsequent second-strand synthesis step. IVT can then use this newly appended T7 promoter and linearly amplify the template dsDNA, producing antisense RNA (aRNA) product. After the IVT reaction is complete, the aRNA is cleaned up using the QIAGEN RNeasy Kit. This protocol for RNA sample purification is based on the manufacturer’s protocol for cleaning up RNA reactions, with minor modifications.

In many experiments it is necessary to section the brain to determine the location of a treatment (lesion or electrode) or to look at the histology of the brain using various staining techniques. Because the texture of the brain is so soft (often likened to soft cheese), it must be "fixed" before it can be removed from the skull. A fixative is a chemical that cross-links the molecules of the tissue, rendering it hard and preserving the tissue. This protocol describes a method for perfusing the brain with fixative (specifically, it describes how to perfuse a rat brain; slight modifications may be needed for different animals). A relatively simple gravity feed and the pumping mechanism of the heart is used to get fixative into the brain. A cannula is placed in the heart, or directly in the ascending aorta, of a deeply anesthetized animal. Blood is flushed out with saline first, and then with a fixative. The choice of fixative is often important if a specific staining technique is to be used, especially in immunocytochemistry, because the fixative can interfere with the staining sensitivity.

In this protocol, the perfused brain of a rat is separated from the surrounding tissue and post-fixed in a formalin/sucrose solution in preparation for freezing and sectioning. Dissection should be done as soon as possible after perfusion to prevent desiccation of the brain. This procedure can also be used to dissect fresh (non-perfused) brains, for example, for Golgi-Cox stain or neurochemical assays.

This protocol describes vector production by transient transfection. The production of retroviral vectors requires a full-length copy of the vector RNA to be incorporated into virions. This is accomplished by coexpressing vector RNA and the viral proteins required for virion formation from expression plasmids. To avoid generation of replication-competent virus, the viral genes are carried by separate plasmids. Generally, the gag and pol genes are on one plasmid, and the viral envelope gene is on a second plasmid. The viral protein-coding regions can be expressed using various promoters to decrease homology and thereby decrease recombination. Because these plasmids do not contain the packaging () sequence, the viral genes are unlikely to be incorporated into virions.

This procedure describes the generation of clonal vector-producing cells that will provide an unlimited amount of unrearranged retroviral vector. The procedure involves transfection of one packaging cell line to generate a vector that is used to transduce a second packaging cell line. The resultant vector-producing clones generally contain a single integrated copy of the retroviral vector, and virus produced from this integrated vector is as genetically homogeneous as possible. Although the vector produced by a given packaging cell line can sometimes be used to transduce the same cell line, the transduction rate is typically low because of receptor blockage by the Env protein made by the target packaging cells. Indeed, this procedure will select for target cells that express low Env protein levels and thus are less resistant to transduction, but at the same time will ultimately produce less vector because of low Env production. Therefore, to obtain the highest vector titers, it is important to use pairs of packaging cells such that receptor blockage is not an issue. In this example, we use PE501 ecotropic packaging cells for transfection and broad-host-range PT67 packaging cells to make stable vector-producing cells.

This protocol is suitable for transduction of many adherent cell lines. The number of target cells transduced can be varied as needed by maintaining the ratio of surface area to volume and using plates/flasks of various sizes. The protocol can also easily be adapted for non-adherent cells using similar vector-to-cell ratios.

Noncycling cells are relatively resistant to transduction with retroviral vectors. Because most immortalized cell lines are actively proliferating, this is not an issue. However, for many primary cells, especially quiescent populations such as primitive progenitor and stem cells, the gene-transfer rate can be particularly low. This protocol describes transduction of primary hematopoietic cells. Two interventions are combined to maximize gene transfer in hematopoietic progenitor cells: (1) cytokines and other growth factors are used to stimulate cell cycling in hematopoietic cells, and (2) matrix proteins such as fibronectin are used to mediate colocalization of target cells and vector.

Scanning electron microscopy (SEM) is used to produce detailed images of surface structures. This article details the steps required to prepare a sample for SEM. In brief, the sample is fixed, dehydrated, subjected to critical-point drying, mounted, and coated with an electrically conducting material such as gold or palladium.

The eukaryotic cell has evolved to compartmentalize its functions and transport various metabolites among cellular compartments. Therefore, in cell biology, the study of organization and structure/function relationships is of great importance. The nucleus contains almost all of the cell’s DNA and is bounded by a double membrane. Inside and adjacent to the inner membrane of the nuclear envelope is the nuclear lamina. It is composed of a fibrous meshwork comprising one or more of three major intermediate filament-like polypeptides: lamins A, B, and C. The outer nuclear membrane is contiguous with the endoplasmic reticulum. Several fluorescent stains are available that label DNA and allow easy visualization of the nucleus in interphase cells and chromosomes in mitotic cells. These stains include Hoechst and 4',6-diamidino-2-phenylindole (DAPI), which are used in this article. Although not as bright as the vital Hoechst stains for DNA, DAPI has greater photostability. One advantage of Hoechst 33342 over DAPI is that the former is membrane-permeant and is, therefore, useful for imaging living cells, because it does not require cell fixation or permeabilization.

This protocol describes chemical mutagenesis of male mice using N-ethyl-N-nitrosourea (ENU), which is the most efficient method for obtaining mouse mutations in phenotype-driven screens. A fractionated dose of ENU, an alkylating agent, can produce a mutation rate as high as 1.5 x 10^–3 in male mouse spermatogonial stem cells. Treatment with ENU produces point mutations that provide a unique mutant resource: They reflect the consequences of single gene changes independent of position effects, provide a fine structure dissection of protein function, display a range of mutant effects from complete or partial loss of function to exaggerated function, and discover gene functions in an unbiased manner. After treatment with ENU, mice are mated in genetic screens designed to uncover mutations of interest. Screens for dominant, recessive, and modifying mutations can be performed.

Desorption electrospray ionization (DESI) is amenable to the study of intact proteins in complex mixtures, including blood or other biological media. Intact proteins can be desorbed and ionized from the surface under gentle (soft) conditions to produce compact conformations of the protein. A procedure for DESI analysis of intact proteins and oligopeptides using mass spectrometry (MS) is described here. DESI-MS is an emerging technique with great promise, but its application range is still being investigated. Therefore, the protocol presented here provides general procedures used for the applications that have been investigated so far. Optimal ion source parameters and surface types may vary depending on the application.

The analytical utility of desorption electrospray ionization (DESI) is such that it can be applied to qualitative proteomics research in the same way as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) methods, although little work has yet been reported in this regard. Because DESI is a surface analysis technique and easily automated, it can be implemented for high-throughput applications, which include the analysis of chromatographic fractions of digested proteins. The analysis of tryptic peptides follows the same protocols as in typical MALDI or ESI methods, except that the mixture is spotted directly onto an insulating surface, allowed to dry, and analyzed directly without adding matrix compounds (as in the case of MALDI methods). The spectral characteristics are similar to those of ESI in that both singly and multiply charged analyte ions are detected. Spectra are highly similar to electrospray spectra of tryptic digests with regard to the overwhelming presence of multiply charged ions of peptides. DESI-mass spectrometry (DESI-MS) is an emerging technique with great promise, but its application range is still being investigated. Therefore, the protocol for DESI-MS analysis of tryptic digests/peptides presented here provides general procedures used for the applications that have been investigated so far. Optimal ion source parameters and surface types may vary, depending on the application.

Desorption electrospray ionization (DESI) allows in situ analysis of biological tissues. The analysis of less abundant protein constituents within a tissue sample often requires the removal of lipid species prior to analysis, similar to the situation with matrix-assisted laser desorption/ionization (MALDI). After removal of lipid constituents, the tissue can be treated with protease to degrade proteins present in the tissue. The tryptic products can be investigated directly from the tissue using DESI. The spectra obtained feature ions of tryptic fragments from abundant proteins present in the tissue sample. The digestion is usually not complete; hence, the presence of missed cleavage sites is typical in the peptides detected. The signal is more stable for longer times than in the case of deposited samples, so the recording of mass spectrometry (MS)/MS data is simple in this case. DESI-MS is an emerging technique with great promise, but its application range is still being investigated. Therefore, the protocol for DESI-MS analysis of tissue sections presented here provides general procedures used for the applications that have been investigated so far. Optimal ion source parameters and surface types may vary depending on the application.

Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein-protein interactions in living cells. This method has been successfully adapted to a variety of expression systems in different organisms. BiFC is based on the formation of a fluorescent complex by fragments of the enhanced yellow fluorescent protein (eYFP) when brought together by the interaction of two associating proteins fused to these fragments. Interaction of these proteins restores fluorescence and allows the visualization of spatial localization patterns of protein complexes. Absence of interaction prevents reassembly of the fluorescent protein and results only in background fluorescence. The specificity of bimolecular fluorescence complementation must be confirmed by parallel analysis of proteins in which the interaction interface has been mutated. This protocol describes the Agrobacterium-mediated transient expression protocol for BiFC assays in Nicotiana benthamiana leaf cells. This method exhibits a high transformation rate (up to 90% of the cells) and allows the simultaneous expression of multiple proteins in single cells. Therefore, this expression system enables colocalization analyses of fluorescently labeled proteins with the formation of BiFC complexes for determination of cellular complex localization. In addition, protein interaction assays in N. benthamiana leaves permit the investigation of protein interactions at different time points of expression, allow analysis of proteins that are normally toxic in protoplasts, and enable comparative protein interaction investigation in epidermal cells as well as in mesophyll protoplasts.