Hi,
Im having a hard time getting good sectioning on my PFA fixed tissue. No matter how I try to cut my sections, brush method or anti roll plate, my tissue is coming apart from the OCT which prevents me from being able to straighten my tissue.
Ive tried using cold or room temperature slides and nothing seems to help make the wrinkles go away since the OCT itself is not attached to the tissue- sometimes it comes off of the entire tissue completely.
I've adjusted the temperature of both the head and the chamber anywhere from -18 to -23 and nothing seems to be helping... Any advice is so welcome and very needed!
Hi Aaliyah Hay
Did you keep the whole brain in gradient sucrose (cryoprotectant) post PFA fixation?
I use the following protocol for my staining.
Anesthetize the animal and perfuse intracardially with 4% PFA. Following perfusion collect the brain and store it in 4% PFA at 4 degree (48hr) or room temp for a few days. After Fixation, the brains have to be kept in gradient sucrose (10, 20 and 30%) until the brain tissue settles below the container. If you are using a cryotome, prepare blocks using OCT medium by snap freezing in liquid nitrogen, cut 40micron sections of the brain and dry the slides for overnight.
Now you can process the slides
1. ice cold Methanol fixation for 10mins
2. PBS wash- 5mins
3. 0.8% Triton-X100 - 10mins
4. 0.2% Triton-X100 + 5% Normal Goat serum (NGS)- 1hr
5. Primary antibody incubation- 1hr (You can check the IF dilution of the antibody)
6. PBS wash- 10mins
7. Secondary antibody- 1hr (Check the species reactivity and dilution)
8. PBS wash-10mins
9. Mount with DAPI containing mounting media
The procedure works quite well for me.
You can try and let me know if you have any further questions.
Thanks
Hi yes I did keep the whole brain in PFA 4% for 48 hours, sucrose 15% around 48 hours and then sucrose 30% around 24 hours. I froze the tissue in OCT in a cryomold held in crushed dry ice and methanol slurry. I need to cut my sections at 10 microns but cutting thicker didnt help mount the tissue as it still splits from the OCT
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