Dear Thuy, I assume that your biopsies are fixed in formalin (either commercial or 4% PFA) and your AR method is using pressure cooker or it's lab equivalent. I would suggest experimenting with microwave induced AR at lower temperature (heating the same citrate buffer pH 6.0 you are using to 80oC in a slide container, inserting slides into heated solution and heating on lowest setting of your microwave. You could test 5, 10, 15 minute MW at the same time, using 3 smaller slide containers. If you work on biopsies - one of those times should be enough - if sections from specimens which were overfixed - you have to experiment). Resting time in the cooling buffer - 30 min. If you are using lab microwave with controlled temperature - that is much easier and you can lower the temperature and prolong MW. Use either commercial non-serum blocking or prepare your own casein block and use the highest dilution of primary antibody which works for 18--24 hours at 4oC (overnight in the fridge). If you have enough spare sections - the best way is to change one aspect of experimental conditions at a time - but if you can't do that - check MW method and serum-free non-specific binding blocking. Good luck with your experiments.

Thank you so much for your answer Maria! I'm also thinking about the option of reducing the temperature of retrieval step but unlucky that our lab don't carry one microwave oven, and the lowest temperature of pressure cooker is 110 degree which I think doesn't have much different effect on reducing background comparing with 120. I also did try 1 hour blocking with normal goat serum room temperature before applying the first antibody.

Tthe microwave is the only option that I haven't went through, so I think I will seek for help from other colleges for that. 

Thanks again for precious comment!

In this case, you could try a water-bath AR (set to 80oC for at least 45 min), which is not as effective as MW, but please try also a non-serum based blocking and diluted antobody overnight at 4oC. If you do not have a waterbath - fill large container with 80oC water, insert glass container with citrate buffer at the same temperature, (Glass container - so it is heavy and stable) then insert slides in a holder). If you try - lease let me know how it worked.

Hello again Maria! With the pressure cooker I did try with and without serum blocking, first antibody 1 hour at room temperature and overnight 4oC but couldn't get good result. Yesterday I tried the waterbath AR as you recommended; however sine our lab don't have the water bath so I put the samples in the glass container with citrate buffer at 66oC and kept in the incubator overnight at that temperature (I read that some lab used this method), followed with 1 hour room temperature for first antibody. Unlucky for me that the result so no different. I'm wondering about too long time in the incubator or should change to same time AR but diluted antibody 4oC?

Dear Thuy,

Beyond all that was said, maybe that strong and (I assume) diffuse background staining wasn't from a excessive AR, but maybe from a inespecific link to terminal sialic-acid residues. If is the case you can block with Neuraminidase. I hope this hint could help.

I agree with Paulo. It is worth to try

Best!