Currently I use Leica BOND Plus slides to mount 5 micon thick FFPE sections of mouse knee joints. These have been fixed in PFA and decalcified in EDTA by experienced researchers, and have been embedded using the following programme in an automated tissue processor:
70% EtOH – 2h
95% EtOH – 2h
100% EtOH – 2h (x3)
Xylene – 2h (x3)
Histowax – 1h (x3)
I dry the sections overnight at 37-40 C on a drying rack, then bake at 65C for 3h and leave the sections to set at RT.
I dewax using the following protocol:
Xylene - 5 min (x3)
100% EtOH - 5 min (x2)
95% EtOH - 5 min
70% EtOH - 5 min
50% EtOH - 5 min
1x PBS - pause point until steamer reaches 99C
Then I proceed with antigen retrieval at 99C in a steamer:
dH2O - 10 sec dip
Tris-EDTA, pH 9.0 - 10 min
1x PBS - pause point at RT.
After the steamer steps, bone and cartilage seem to have fallen of the slide, folds are introduced, etc.
As for the other tissues, the muscle seems to have some folds, while the marrow is mostly intact and does not detach.
I've tried in parallel a different buffer (Citrate buffer, pH 6.0), an overnight 60C incubation in a water bath, as well as a pressure cooker. In all cases the same effect is observed, including if I just stick my tissues in water at 99C for 10 min. This leads me to believe the bone and cartilage tissue is not sticking properly to the slides.
Do you have any tips/recommendations on what I could be doing wrong?
Hi Jordan,
I did histology of bone and cartilage of rat and human. Here are some papers. I am sure you can figure out good information relevant to you from these my publications. You can e-mail me any question. My antigen retrieval methods were some different kinds too. I did not find sections falling from slides. Good luck
Thanks a lot, Subhash C. Juneja !
The antigen retrieval step I've been trying to optimise is part of a larger protool for digital spatial profiling by Nnostring, they have insisted on the Tris-EDTA method, but it might be that for my tissues an enzymatic antigen retrieval is best. The protocol includes a proteinase K step, but at a milder concentration, I will see what I can do to test if this step is sufficient, perhaps trying a few different concentrations.
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