Staining of Drosophila Tissues with Antibodies
This protocol was adapted from “Histological Techniques for the Drosophila Eye. Part I: Larva and Pupa,” Chapter 12, in Drosophila Protocols (eds. Sullivan et al.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2000.INTRODUCTION
Staining Drosophila tissues with antibodies is generally straightforward. Ideal concentrations must be determined for each antibody: generally, dilute monoclonal supernatants 1:1 and ascites or sera 1:250 to 1:5000. If double-labeling, confirm that the secondary antibodies used will not recognize each other. Cross-reactivity can be avoided if secondary antibodies are raised in a single species. Fluorescent double-labeling generally does not work if both primaries are raised in the same species unless the primaries are directly conjugated to fluorochromes. If using primary antibodies raised in the same species, the use of HRP-conjugated antibodies is recommended. Double-labeling using DAB requires that the reaction product for one of the antibodies be intensified. This process is sensitive and should be used to detect the less abundant antigen. Similarly, if one of the primary antibodies gives a high background or is widely expressed throughout the tissue, this signal should not be enhanced. If one antigen is nuclear and the second is either cytoplasmic or on the cell surface, it is better to intensify the nuclear signal; the enhanced product can obscure details recognized by the cytoplasmic/cell surface antibody. If one antigen is less stable (e.g., extracted by detergent), this antigen should be detected first.
