Hello everyone, I just started learning IHC staining and I have some frozen sections to practice with in a 96-well plate. I'm having trouble mounting the sections on the slide, a colleague told me to use just 1xPBS to float the section (she uses PBS + triton, but told me I shouldn't begin with that) and then mount it using a brush on the slide, but it's much harder than it looks, and the sections always sink and I screwed many sections because they are so delicate. Do I need to change the solution? I watched many videos for the technique, but I don't know if there's a better solution, or any tips generally.
Hi Ahmad El-Hamamy
Personnally, I start by putting all the sections I want to mount in a petri dish, filled with buffer. I generally use 50mM Tris-HCl (pH=7.5) for that purpose, in order to avoid salts, but it can also be 1X PBS if you want.
All the sections should be floating in the petri dish. Then, I place a thick brush right below a section, then move the brush up, until it's out of the buffer. Now, the section should be rolled up on the brush. Finally, I flip back this brush onto the slide, without pushing too hard, not to crush everything. With a second brush (a thin one), I gently help the section to "unroll" on the slide. I don't know if it's clear enough, it's quite hard to describe the whole process.
This works well for free-floating, 30µm, frozen sections from mouse brains. For thinner sections, it's of course more difficult. The quality and "level" of fixation also has an impact. The less fixed the tissue is, the more fragile it will be.
Finally, I strongly encourage you to use gelatin-coated slides. It will facilitate a lot the handling of sections on the slide, as you will be able to use your brush to move them around without damage. I have used regular slides before, and mounting the sections was a nightmare, very long and very difficult. When I switched to gelatin coated slides, it was a life-saver!
Vincent Simon
Thanks a lot for your detailed answer, very helpful. Unfortunately my samples are much thinner than that I believe, I don't know exactly how thin, I will ask, but they seem they will never withstand all that rolling and flipping and unrolling, they are just too fragile!
Today is my first day of doing that, so maybe my poor technique is the reason. I will try as much as I can to improve, and see if I can get my hands on those gelatin-coated slides. Thanks a lot, appreciate it.
I would suggest using a small artist brush to pick up the section then gently transfer it to a drop of dH2O on a slide and allow the drop to gently evaporate at 37C. Make sure the slide is coated with a very thin film of albumin fixative. Stay away from salt buffers as they will crystallize as the drop evaporates.
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