Protocol

Staining Whole Mouse Embryos for ß-Galactosidase (lacZ) Activity

This protocol was adapted from "Techniques for Visualizing Gene Products, Cells, Tissues, and Organ Systems," Chapter 16, in Manipulating the Mouse Embryo, 3rd edition (eds. Nagy et al.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

Whole mouse embryos are stained for ß-galactosidase (lacZ) activity using X-gal.

RELATED INFORMATION

A protocol for Staining Frozen Mouse Embryo Sections for ß-Galactosidase (lacZ) Activity is also available.

MATERIALS

Reagents

Detergent rinse

Embryo staining solution

If staining is to be performed for more than 1 hour, add Tris-Cl (pH 7.3) to the staining solution to a final concentration of 20 mM.

Ethanol (optional; see Steps 4 and 5)

Fixative for staining intact embryos, freshly prepared

Mouse embryos (13 dpc or older) or pregnant mice (for embryos 15 dpc or younger)

Nuclear fast red (optional; see Step 5)

Paraformaldehyde (0.4%, w/v) in 1X PBS (optional; see Step 2)

Xylene (optional; see Step 5)

Wax (optional; see Step 6)

Equipment

Dissecting tools

Incubator, preset to 37°C

Microscope (optional; see Step 6)

Pasteur pipette (optional; see Step 2)

Tissue culture dish, 24-well (optional; see Step 2)

METHOD

1. Dissect embryos free of their extraembryonic membranes.

i. For embryos 13 days or older, slice them in half sagittally with a razor blade to facilitate penetration.

ii. For embryos from 15 dpc to birth, it is advisable to perfuse the pregnant mouse.

2. Fix the embryos in fixative solution for best results. When examining many embryos at one time, place them individually in wells of a 24-well tissue culture dish and aspirate the fixative solutions with a Pasteur pipette. The fixation time depends on the size of the sample:

i. Fix tissue culture cells for 5 minutes.

ii. Fix early postimplantation embryos for 10-15 minutes.

iii. Fix embryos up to ~13 dpc for 15-30 minutes.
If lacZ histochemistry is to be followed by antibody staining, a 0.4% paraformaldehyde fixation solution can be used instead.

3. Rinse the fixed tissues in detergent rinse three times for 15-30 minutes at room temperature.

4. Incubate the samples in embryo staining solution. The staining time depends on the size of the sample and the level of bacterial ß-galactosidase activity:

i. Incubate small samples in embryo staining solution at 37°C for 1-3 hours in the dark.

ii. Incubate older embryos in embryo staining solution at 37°C for 4-5 hours or longer in the dark.
At this stage, embryos may be stored at 4°C in 70% ethanol. To image whole embryos, please see Imaging X-gal-Stained Mouse Embryos. To section embryos and observe lacZ expression at the cellular level, proceed to Steps 5-6.

5. After staining, dehydrate the embryos through a graded series of ethanol (70%, 90%, 95%, and 100%, prepared in xylene).
Do not leave the samples in alcohols and solvents longer than necessary because they may leach out the reaction product. Nuclear fast red can be used as a counterstain.

6. Embed the embryos in wax, section them (see Sectioning Mouse Embryos, and view them under a microscope.
When sections are viewed by dark-field microscopy, the lacZ stain is pink and the contrast with surrounding tissues is good; therefore, no counterstaining is necessary.