Hello everyone,
I am performing IHC for von Willebrand Factor on femurs and tibiae of 22-week-old SD rats. The bones are decalcified in EDTA and paraffin embedded.
After antigen retrieval, however, the bone in my sections is coming off of my slides.
My slides are polylysine coated Thermoscientific SuperFrost slides. Antigen retrieval is 4x 5mins in a 950W microwave, immersed in 0.01M citric acid buffer pH6.
I am quite sure that the boiling of the buffer is what is causing the bone to fall off the slides.
Is this correct? Do I need to employ a more gentle antigen retrieval technique? Would a waterbath at 60C for 1hour get similar results to a microwave?
Any help on the matter would be much appreciated.
Thank you for your time.
What thickness are the sections? Thinner would be better 5-7 um if possible. 2 suggestions: 1) incubate USS at 60C overnight , we have found that mitigates section loss with high adhesive slides. We are not performing AR in MW, but using automated platform with max temp 95C. 2) Water bath is probaoly best but you can still do in the microwave just use lower power settings (% of power output). suggest test with positive control to optimize time frames and temperatures
Luis Chiriboga ok thanks very much for your help! I'll try and source a waterbath.
I agree with Flynn Lachendro about baking your slides overnight. Also, try the antigen retreival with Citrate Buffer at 37 degrees celsius for 20 minutes if you are still having issues. Also, you can try a different antigen retrieval method such as Trypsin/EDTA at 37 degrees celsius for about 20-30 minutes. I have found this to work well and is much gentler on your samples.
Thanks very much Heather Wanczyk . Would this short incubation time still work for bones which have been fixed in 10% formalin for 48hrs?
Dear Flynn,
what we usually do with tissues that tend to come off is to immerse the polylysine coated slides in polylysine and let them dry. This additional coat is usually very helpful. Moreover, we usually perform the microwave antigen retrieval at lower power, that is more gentle (200w, 15min) after pre-heating the buffer for 2 minutes at the maximum power.
I hope this can help.
Good luck!
Flynn Lachendro, try it out. It should work as long as the bone is sufficiently fixed to the slide.
Also, see recommendations from this website to impove the adhesion of your samples to slides. rhttp://www.ihcworld.com/_technical_tips/section_falloff_tips.htm
Hi Flynn, how long you decalcified. when my colleague did for 6 weeks in EDTA he faced lot of problems while cutting. generally more literature is available on mouse bone studies. when I did I decalcified for more than 4 months in EDTA. EDTA takes lot of time. you can reduce time with other agents (see attached). as Luis suggested thickness of sections should be around 6uM hope this helps ...
For a general reference, see Gambella, A. et al: 2017 Section detachment in immunohistochemistry: causes, troubleshooting, and problem-solving.
Unfortunately they did not specifically look at decal solutions
Venkateswara Rao Alapati, thank you for uploading the paper, it is very interesting. I'm also facing some troubles with mouse femur sections, that can easily detach from the slides. In the past I used other acid-based decalc. methods, but having a lot of troubles in preserving antigens. I'll give a try to the 6% TCA method, too. I'm currently using EDTA to decalcify bones (0.5M, pH 8.0, for 5-7 days at 4°C). By the way, I got the very opposite impression, in my experience, since I was incubating mouse bones for 4-6 weeks, and the sections (cryosections) were falling off the slides very easily. Just by incubating with the antibody solution, without any need for antigen retrieval. Now I'm doing the same for just 5-7 days at 4°C, and things got much better.
Flynn Lachendro have you ever tried to use decalcified frozen bones? This will overcome the need for AR. Obviously, this doesn't solve your problem with the paraffin-embedded bones that you already have.
Luis Chiriboga raised a good point, actually the problem of doing AR in the microwave is that you can not control easily if and when the solution is boiling, and the bubbles that are produced can often detach sections from the slides. Another way of doing the AR would be to use a pressure cooker, but using a 95°C water bath is actually a good suggestion.
Heather Wanczyk, I'm pretty sure that an antigen retrieval with citrate buffer at 37°C is almost useless, as at this temperature you can't achieve a sufficient de-crosslinking. Take a look at this paper: Article Molecular mechanisms of antigen retrieval: Antigen retrieval...
Serenella Silvestri, thank you for your suggestion, I'll give a try, too.
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