A method for the demonstration of NADPH-diaphorase activity in anuran species using unfixed retinal wholemounts.

The presence of NADPH-diaphorase enzyme has been previously revealed in fixed mammalian retinal tissue (Sagar, 1986). Fixed retinae of Bufo marinus and Xenopus laevis failed to yield selective staining when reacted for NADPH-diaphorase. Satisfactory staining of retinal neurons was attained when the histochemical reaction was carried out in unfixed retinal wholemounts. The applied method included the following steps: 1) Dissection of the fresh retina and the separation of the neural retina from all other coats of the eye ball, including the vitreal tissue; 2) pretreatment with 300 mM sucrose in phosphate buffer; 3) incubation with NADP, malic acid and nitroblue tetrazolium in phosphate buffer (pH 7.6); and 4) fixation of the tissue in 10% buffered formaldehyde overnight followed by whole mounting. For control, fixed and unfixed rabbit and human retinae were also reacted for NADPH-diaphorase according to the above method. In these species specific staining was achieved only with fixed tissues. The possible implications of revealing NADPH-diaphorase enzyme activity in fixed mammalian and non-fixed anuran tissues are discussed.