I m working on coronal section of swiss albino mice strain brain for studying some aspects of immunofluorescence on hippocampal portion, could anybody help me with
How much thickness of coronal section of mice brain should be cryosection it using cryostat?
or how much thickness of coronal section, I should cut with cryostat so that it can be placed proper on APTS coated slides and process further for imaging.??
Yesterday, after washing in pbs to remove freezing medium. All sections taken on slides got removed.
Please help me if somebody have experience and share there best protocol cum experience to get out this problem.
Thanks
Regarding the microns you can go up to 20 microns. I wouldn't recommend more than that. It can depend on the positiveness of the antibody you are using. For better or stronger signal detection use section of 20. If not, use 10-15 microns.
I also recommend Poly-L Lysine slide (purchased). You can coat your slides cheaper on the lab, but it can give you false positive results or background staining.
Hussein Mansour, Ricardo Camarinho: Thanks for sharing your experience.
I have done exactly the same steps as you told and used the Poly-L Lysine slide (purchased) and the coronal section thickness was about 12uM. So after sectioning it showed the good adherence over the precoated slides. then encircled the good sections with hydrophobic pen on slides. However, during washing step of PBS + Triton X-100 (for removing the cryostat freezing medium) the adherent sections on some precoated slides were getting removed from slides even without any shaking.
So, I kept it as stagnant for some minutes. After that blocking with 5 % BSA was also causing the same thing of removing the sections from slides. We kept it carefully with primary antibody with overnight incubation.
Please tell the way to rid off this removal of sections even on precoated slides....
Thanking you
Hi Mohit Kwatra,
For better tissue sample adhesion on slides, you again coat/use a drop of diluted 3- aminopropyltriethoxysilane and dry for 10-15 min and use this side.
I did during my experiment. You can try.
Thanks
Hi Mohit,
I think there are two things you should consider
1. Section thickness: I cut my cryosections (the tissue was 4PFA fixed, sucrose cryoprotected) depends on what I plan to do: if I plan to do a quantitative study, I cut 30um so I can get more signals; if I want to show the good morphology, I cut 7-15um, then I can get low background, crisp signals.
2. Problem of section falling off slides: I usually use "superfrost plus microscope slides" from Fisher Scientific. It is very good for frozen sections. Since I started to use it, I have never experienced the section falling off problem that often happened on my slides before. Another, make sure you dry your sections after you cut them (I usually cut the sections, dry them in room temperature overnight or dry in 37 degree for 1 hour), after drying it, immediately start the staining procedure.
Good Luck,
Chunfeng
Thanks Ricardo Camarinho and Chunfeng Tan..
I will try your valuable suggestions :)
Hi Mohit!
For imunohistchemical/immunofluorescencent staining of frozen brain (rat) tissue we use 10 µm brain sections. The brains are removed, quickly frozen on dry ice and sectioned in a cryostat at -20 °C. The sections are adhered to the microscope glass slides coated with 0.01% solution of (poly) L-lysine (Sigma). The sections are allowed to air dry (for an hour or so, room temperature) and then stored in a freezer at -20 or -80°C (vacuum-packed). For imunostaining brain slices are fixed with cold methanol (-20°C) for 30 minutes. Blocking solution contains 4% normal serum, 1% bovine serum albumin (BSA) and 0.1% Triton X-100 (all from Sigma) in potassium phosphate buffer saline (KPBS).
We have never experienced removal of the sections from the slides during immunostaining.
Hope this helps!
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