Stomach tissue might have a specific pH depending on the digestive time it was taken. Then you have to control that number and equalize optimizing all pHs: antigen, buffer and tissue.
Depends mostly on your antigen and is not always pH. You have to optimize almost everything, especially if you don't have some preliminary testing.
Optimize. I have come to find that the pH is the critical point along with the timing. The buffer composition is not as critical in the end at least for my applications. I have even tested used dH2O with hydrochloric acid to pH to the desired point and had fine results (human brain, formalin fixed, paraffin embedded) My routine is the 10mM citrate buffer pH 6.0 and microwave 8-10 minutes until "simmering". I use one liter of solution in a plastic container. (we do a LOT of slides at a time) Slide load is important too, watch to make sure of course you don't loose liquid or over cook. I take out the bath and run under cool tap water immediately. I like to see that the section seems very slightly translucent than when I started otherwise the cells will look damaged.
One qualitative observation: a freshly cut paraffin section will seem to be MORE sensitive to the AR than one that has sat around for let's say a few days. Why I don't know unless that gives us a clue to remaining moisture in the paraffin block/section?
And a P.S. if the method says for you to use microwave then DON'T boil it in a beaker. Someone that I gave the microwave retrieval method to did that, called me and said it didn't work and digested the sections too much. When they finally said it was boiled in a beaker and not in a microwave I knew what happened. All they said it was the same idea. Not exactly. The microwave energy is thought to help accelerate the breaking of the bonds caused by the formaldehyde.
For antigen unmasking, in ileum sections I heated them in sodium citrate buffer (pH 6.0) in a microwave (two cycles of 5 min at 800 W) and I had good results.
you can give 30 minutes in pressure cooker or 15 to 20 min with microwave. but i would suggest to standardize your own protocol this can be helpful in defining ranges.
you can give 30 minutes in pressure cooker or 15 to 20 min with microwave. but i would suggest to standardize your own protocol this can be helpful in defining ranges.
For antigen unmasking, in pig and rat stomach sections I used with good results sodium citrate buffer (pH 6.0) in a microwave (two cycles of 5 min at 800W).
The answer depends on the antigen you wish to examine. It may be that enzyme digestion gives optimal results. Test with no retrieval, enzyme digestion, and citrate and EDTA heat retrieval. As to time, that is something you would also need to experiment with but I would start with 5 min enzyme @ 37C, and 20 min @ 100C for your buffer retrieval.
Your results will also be affected by which type of detection system you are employing and whether you are performing the IHC manually or on an automated platform.
You must optimize and then validate any new antibody you wish to look at to YOUR conditions and not what someone else is doing in their lab.
Thank you so much. I have already standarized in my lab. I used citrate buffer (pH 6.0). Preheated retrieval solution to 92-95C in water beth. I placed polypropylene couplin staining jar. Immersed slides into preheated retrieval solution for 10 min. After the incubation is finished, remove the couplin jar with retrieval solution and slides from the water bath, and let it cool to RT.Then gently rinse the slides with deionized water and then with PBS, 2X for 5 min (Note: Because tissues may be loosened after the retrieval procedure, avoid vigorous rinsing to prevent detachment from the slides).
Hi, we use IHC almost every day in our lab. Like others said, duration of antigen retrieval depends on your antibody. Steps are below for you to follow
1, Preheat between 50-70degree Celsius for 5 mins.
2, Put your deparaffinised slides in those preheated citrate buffer.
3, Continue heating with low pressure between 10-30mins. You can try optimisation by choosing 10mins, 20mins and 30mins. Choose the best incubation time for you.
Adding tween 20 (0.05%) into the citrate buffer can be a game changer.
Best Wishes
Thank you very much for your response. As I mentioned I have already optimized the incubation time. Thanks again.
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