Tissue fixation and immunohistochemistry protocols have been described in detail previously.10'11'20 Briefly, carbodiimide fixations involved transcardial perfusion with 2 to 5 times the body volume of an aqueous solution of 6% EDAC and 5% DMSO containing 1mM N-hydroxysuccinimide. The fixative solutions were heated to 37oC before initiating the perfusions, because the coupling reaction proceeds significantly faster at higher fixative temperatures. Post-fixations are done in 4% freshly depolymerized paraformaldehyde, or 10% neutral buffered formalin for 24 to 48 hours. For invertebrates or other animals that are difficult to perfuse transcardially, tissues were fixed by removal and rapid immersion in the standard EDAC fixative containing 5% DMSO and 1mM N-hydroxysuccinimide for 20 minutes, followed by further fixation in buffered formalin for at least 24 hours.

Immunohistochemistry was done by the avidin-biotin complex method (Vectastain Elite; Vector Labs, Burlingame, CA). Primary rabbit antibodies were diluted appropriately in 2% normal goat serum (NGS) and incubated with tissue sections for 48

to 72 hours at room temperature with constant rotary agitation (with 0.1% sodium azide as preservative). The secondary antibody and HRP-labeled avidin-biotin complex solutions were incubated with sections for 60-70 minutes each, and after washing were developed with a nickel and cobalt enhanced diaminobenzidine chromogen system (Pierce Biotechnology, Rockford, IL).

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