Jun 12, 2023

Public workspaceAFOG V.1

DOI
dx.doi.org/10.17504/protocols.io.bp2l6xkrzlqe/v1
  • 1iMM
Joana G Antunes
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.bp2l6xkrzlqe/v1
Protocol Citation: Joana G Antunes 2023. AFOG. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6xkrzlqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: June 12, 2023
Last Modified: June 12, 2023
Protocol Integer ID: 83248
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.
Protocol materials
ReagentDistilled Water
Step 5
ReagentBouins solution
Step 6
Preparation
Preparation
1h 30m
1h 30m
Warm the cryosections from the -80º freezer toTemperatureRoom temperature Duration00:30:00 ;

30m
If gelatin embedded tissue, incubate in Reagent1X PBS (Phosphate-buffered saline ) Duration00:15:00 Temperature37 °C ;

15m
Fix the slides in 4% PFA for Duration00:05:00 TemperatureRoom temperature
Note
Apply the PFA directly to the slides (PFA 4% can be reused, store at 4ºC)

5m
Wash 1x in Reagent1X PBS (Phosphate-buffered saline ) Duration00:05:00

5m
Wash 2x in ReagentDistilled WaterContributed by users Duration00:05:00

5m
Heat ReagentBouins solutionContributed by users Temperature60 °C Duration00:30:00

30m
Staining
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;