4.1. Immunocytochemistry and NADPH-Diaphorase

Immunolabeling of the pineal organ with a highly specific antibody against cGMP revealed predominantely stained photoreceptor-like cells. Double-labeling of the same sections with an antiserum against S-antigen, a specific marker for retinal and pineal

light dark light dark adapted adapted adapted adapted +SNAP +SNAP

Figure 1. Semiquantitative measurement of the intensity of cGMP-immunofluorescence after treatment of the light- and dark-adapted pineal organs with 100 |iM S-nitroso-N-penicillamine (SNAP) for 5 minutes. The intensity of FITC-immunostained slices was determined from photographs using an image analysis system RAG 200 (Biocom/France) and expressed in % area of the entire pineal area with an optical density >l00 (arbitrary units). **P < 0.001 SNAP treated pineal organs vs coresponding groups (light-or dark-adapted); *P < 0.01 dark-adapted vs. light-adapted group.

photoreceptors, showed that most cells that are immunopositive to cGMP represent true photoreceptor cells.

Dark-adapted pineal organs showed a stronger cGMP-immunoreaction compared to light-adapted organs. Preincubation of pineal organs with the nitric oxide (NO) donor SNAP resulted in a strong increase in cGMP-immunoreactivity. These results were confirmed by a semiquantitative evaluation of FITC-immunostained slices using an image analysis system RAG 200 (Biocom, France) (Figure 1).

Staining of the same section of the pineal with the NADPH-diaphorase technique demonstrated that NADPH-diaphorase-positive cells were located in close proximity to cGMP- and S-antigen-positive cells.

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