Hi,
I recently tested an antigen retrieval protocol using a citrate buffer on frozen slides. I obtained a clearer staining, and I'm willing to use this technique routinely.
However, I think that my protocol could be more efficient and maybe quicker, and I thought that perhaps, skilled scientists on Researchgate could help me to do so.
I use cryostat sections of mouse brain (15-20 um thick), and here is the protocol I tested:
For the citrate buffer preparation:
- 2,94g of tri-sodium citrate (dihydrate) in 1L of distilled water, mix
- pH set to 6.0 using HCl
- 0,5 mL of Tween-20, mix
I rehydrated the tissue section, immersing it 2 minutes in ethanol 100%, then 1 minute in ethanol 95%, 1 minute in ethanol 80%, and I rinsed it in distilled water.
After that, I put the slide on top of a dry water-bath set at 95°C, with the tissue section immersed in the citrate buffer for 10 minutes. I added some buffer during these 10 minutes to avoid tissue drying.
I let the slides cool down for 10 minutes with the buffer still on, and rinsed 2x1 minute with PBS.
Then I immersed the sections in my blocking solution (PBS and distilled water with 0,3% triton X100, 3% BSA, 5% goat serum) for half an hour, and went through my usual protocol for immunohistochemistry.
I hope that I have been clear and that some of you will be kind enough to help me !
Hi Fanny, if you got good results you can work with this protocol. If it is ove-r or underexpressed make a delution raw of primary antibody to find out which is the optimal delution for your antibody under those circumstances you like to work with. Positive controls that means tissue which exactly expresses the eptitope of your interest will be helpfull to establish the technique as well. Here is my protocol:
25 µm thick sections, PFA fixed and mounted on superfrost plus slides were rinsed in PBS 3 x 10 min; 4 x 5 Min in 0,01 M Citrate buffer pH 6 in the 800w microwave; cool down in Microwave for 10 min; cool down outside microwave for 10 min, 0,0 M Citratrate buffer pH 6 handwarm for 10 min; 3 x 5 PBS; start your IHC protocol. Good luck!
HI Fanny, I agree if your results are "good", then all you can do is stick with this protocol, or try another to see if results are "better"! I'm confused about the dehydration step... are you starting with freshly sectioned frozen tissue? If so, it is already hydrated (well, it is not dehydrated).
Also, I really recommend an improvement in the heating to avoid drying. Many use a microwave to heat the buffer. I prefer a home-kitchen rice steamer. Heat the buffer in a microwave while the steamer is heating up. Transfer the buffer to a metal or glass staining dish, and insert the slide carrier into the hot buffer. Steam for 10-25 min. Cool.
Hi Ute and Charles, thank you for your answers,
Ute: I already made some titration studies and determined the best dilution for each antibody, but I did it before trying out the antigen retrieval protocol. Do I have to re-titer the antibodies? (I fear yes..). Also, I'm working on getting my positive controls.
Charles: I don't stain right after the cryostat, because I cut slices of pretty much the whole brain, so I keep all of them at a -80°C freezer. I'm also a bit confused about this rehydratation (not dehydratation) step; I built my protocol using different ones found online, and I think this step may belong to a protocol made for parrafine embedded tissues. I think that I'll just rinse the slices in distilled water for my next attempt, then proceed with the citrate buffer.
For the heating step, I tried to use a microwave, that was way too powerful (even at its minimum), and the citrate buffer was boiling all over the place. The lab doesn't own a steamer or pressure cooker, so I had to figure an alternative...
Fanny, then I'd use a hot plate to heat the buffer to almost boiling. Use care! The steamer just keeps the buffer hot, but you could keep it on a hot plate. People use steamers and pressure cookers because of the dangers of hot liquids on the hot plate, and for better control. A metal slide staining dish is safer from a breakage point of view.
Regarding the rehydration steps, you are currently going from a frozen (hydrated) slice directly to 100% ethanol - that would be a shock to the tissue. I agree you should omit the ethanol steps (until after staining).
Hi Fanny,
You used cryosections, Am I right? Do you pre-fixed your samples in some aldehyde before or after cryopreservation? If not, probably you don't need a antigen recover. Another point, the cryopreserved samples don't need to be rehydrated, only is need when you dehydrate your sample before preservation.
good luck
Thank you Charles and Rodrigo! I wasn't expecting so much help!
Charles: I didn't think about the hot plates! I'll make some experiments to identify the highest temperature I can reach without danger. (this is what I used to heat the slides up in my first experience: http://www.grantinstruments.com/btd-dry-block-heating-systems/). I share your feel about the rehydratation; I won't do it next time. Thank you!
Rodrigo: I perform an intra-cardiac perfusion with 50 ml paraformaldehyde 4%, then the dissected brain is immersed in PFA 4% for 16-24 hours, and finally immersed in sucrose 30% for minimum 24 hours. After these steps, I do the cryosections, which I keep at -80°C until I use them for the stainings. I think that in my case, the antigen recover is useful since I obtained a clearer staining of the slices. And yes, as I was saying to Charles, I will skip the rehydratation step! Thank you very much.
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