Hi.
I am doing immunohistochemistry staining on mice tissue. We perfuse the mice and place the brain in 4% PFA O/N then switch to 48hr in 30% sucrose (or until the brain sinks). However I am finding holes in my samples. Some samples have it worse than others. More particularly, it seems like the cells are nonexistent in my hippocampus region (see photos) but the DAPi is still stained. I see some cells, but not a whole lot in other parts and I'm wondering if those holes were supposedly the cells or just improper fixation
I embed the sample in OCT then place it in -80C until I need to use it (i also let the sample sit out for 1.5 hour before slicing)
Here is also my protocol:
1. Wash in 4% PFA for 9 minutes
2. Wash 3x in PBST 5min
3. permealize slide with .1% Triton and PBS for 10 min
4. Wash once with PBST
5. Appl primary and incubate O/N at 4C
8. Wash slide 3x with PBST
9. Apply secondary and incubate for 1 hr
10. Wash slide 3x with PBST
11. Add a drop of DAPI
12. Add coverslip and seal slide with nail polish
Any suggestions on what's happening?
You do not mention what solution you use for your perfusion. It should be 4% PFA.
What flow-rate do you have for your perfusion? This needs to be sufficiently high (~20ml/min) to expel the blood and for rapid infusion of fix.
Have you tried shorter post-fixation times - this may help?
Why wash in fix for 9 minutes (Step1)? To fix sections on to slides? Why not try using free-floating sections?
Why rinse in PBST (steps 1, 2, 4 and 8)?
Do you use a blocking serum?
What is the penetration of immunolabelling like? Is it limited to the surface of the tissue, or does it go deep into the section? How consistent is the labelling as you go deeper into the section?
I assume you're using a confocal microscope to scan? If so, your DAPI labelling looks saturated - check your saturation levels for all channels.
Do you use tail vein terminal fixing or do you have permission on you project licence to perfuse the carotid artery?
Hello
Kindly look at the link in below will help you
https://biomedizin.unibas.ch/fileadmin/user_upload/biomedizin/core_facilities/histology/Guides/Good_Histology_Practice.pdf
Good Luck
Hi Huyen
You did'nt reveal here which staining is this. It seems this is fluorojade-c (FJ-C) staining that stained specifically dead neurons. In FJ-C staining normal neurons appear like holes in brain tissue as I have already expereinced it. If you are performing any other specific staining it may be a issue of tissue processing as you are facing. In my opinian perform simple H&E of the same tissue and look into the matter itself. All the best!!!
I've seen holes in tissues stained with H&E when they are not properly dehydrated. I do not know if this could be the same problem with your preparations. Perhaps you need to extend the dehydration steps for a longer time since these tissues are previously exposed to a high concentration of sucrose that may be keeping water for a prolonged period of time as compared with other tissues.
Podrías congelar tus muestras en isopentano enfriado en nitrógeno líquido, así evitarás una congelación tan brusca de tu tejido y con ello evitas también la formación de cristales intratisulares. Creo que eses "agujeros" que ves podrían estar relacionados con ese cambio tan brusco de temperatura en la congelación.
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