Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot4526

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Antibody Addition to Drosophila Specimens and Detection Using Enzyme-Linked Reagents

Ed Harlow and David Lane

This protocol was adapted from "Staining Tissues," Chapter 6, in Using Antibodies by Ed Harlow and David Lane. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1999.


INTRODUCTION

Enzyme-linked reagents give excellent sensitivity and use a simple light microscope for detection. A range of enzymes is available, but for staining in situ, horseradish peroxidase will suit most needs. Diaminobenzidine (DAB) is one of the most sensitive substrates for horseradish peroxidase. It yields an intense brown product that is insoluble in both water and alcohol. It can be made more sensitive by adding metal salts such as cobalt or nickel to the substrate solution. The reaction products are slate gray to black, and they are stable in both water and alcohol. DAB/metal staining is compatible with a wide range of common histological stains.


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