Hey everyone,
i perform immunofluorescence imaging on 100µm brain sections.
My protocol for my free floating staining.
Fix the slices in PFA 4% overnight
Wash 3x times in PBS
Blocking for 1 hour with 10%NGS and 0.5% Triton X-100
First antibody overnight
Washing 3x times PBS
Secondary Antibody 2hour at room temperature
Washing 3x times PBS
Covering
I would like to perform colocalization experiments, but the red channel doesnt work very well.
1. The signals are weak
2. The signals are inconsisten
3. The background is high
4. Only some parts of the slice are stained
5. Weird interactions with the staining in the green chanel.
Does anyone has some hints how i can improve the staining in my red channel.
Thank you for the help
seems like there may be pretty easy solutions:
i've used this method for several years and based on everything i've seen you won't be able to stain your tissue with 100% penetration of primary and 2ndary antibodies on 100 um sections. In fact, i've used exactly the same protocol (except for 0.1% TX100) and i noticed after doing confocal stacks that sections thicker than 30 microns look like a "sandwich" of 2 fluorescent sheets with dim area in between (where the antibodies could not penetrate). My suggestion: reduce the thickness of sections to 25-30microns, max 40 microns.
Additional suggestion: your antibodies might be old, if you're storing your 2ndary antibodies at 4C. If not, then the primary reason is the thickness of sections. I will be happy to discuss further. The method produces beautiful 3D images of brain, e.g., see Article Long-Term, Stable Differentiation Of Human Embryonic Stem Ce...
Once you work out the problem, i recommend dehydrating thick sections after mounting on + slides, using ethanols and xylenes, this will compress thick section (e.g., 30um section becomes 12-15um thick), this is great for imaging and also removes lipids. you mount of course in xylene-based DPX mounting medium. Fluorescent signal is not impacted by xylenes (at least Cy2,Cy3, Cy5). Good luck
Please, you look these protocols,
https://bio-protocol.org/prep98
https://www.peprotech.com/en/protocols-Immunofluorescence-General-Animal-Brain-Tissue
here the main issue sounds to be with the thickness of the tissue you are working with.
For immunostaining, 20-25µm thickness works well with most antigens.
Do PBS washes with triton, i would do 2%. Do overnight incubation of antibodies in RT with agitation and block with Sudan B. Do all incubations with agitation
How could you cut so thick slides? Is it really possible to acchive 100µm with cryostat?
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