AFOG
Joana G Antunes
Abstract
Staining technique largely used to diagnose ischemic cardiac lesions and kidney diseases, in particular the regeneration or scarring of the lesion. The selectivity in this method is due to the different affinity degrees between dyes and the macromolecules of the tissue. A key role is played by phosphomolybdic acid, which acts as a binding agent between tissue structures (collagen, fibrils, cell membranes) and aniline blue (amphoteric dye). Orange G, which is the second component of an AFOG solution, has no affinity for phosphomolybdic acid, and this is why it is used to stain all remaining structures. Acid fuchsin, conversely, dyes any protein deposits selectively.
Steps
Preparation
Warm the cryosections from the -80º freezer toRoom temperature 0h 30m 0s ;
If gelatin embedded tissue, incubate in 0h 15m 0s 37°C ;
Fix the slides in 4% PFA for 0h 5m 0s``Room temperature
Wash 1x in 0h 5m 0s
Wash 2x in 0h 5m 0s
Heat 60°C 0h 30m 0s
Staining
Place the slides in Bouin’s solution for 2h at 60ºC;
Continue to stain in Bouin’s solution for an additional 1h at room temp (can be reused 3 times);
Wash slides with dH2O 5-6 times for 5 min until the water comes out clear;
Rinse slides in 1% Phosphomolybdic acid for 5 min (can be reused 2 times);
Rinse slides in dH2O for 5 min;
Rinse slides in AFOG staining solution for 10min (can be reused);
Rinse slides in dH2O for 2 min;
