I usually fix cells with Formaldehyde 3.6-4% at 4 degrees, I was having a poor staining, may this be due to the crosslinking induced by formalin? additionally, what is the consequence if reducing the wasing after incubating with formalin?
Hi Zahraa Fakih
Following plating cells in PDL coated coverslips, I follow the below protocol.
1. 4% ice cold PFA- 10-15mins in room temp (needs to be optimised depending on the cell type).
2. 0.1% T-X100- 10mins
3. 5% NGS blocking- 1hr
4. Primary antibody incubation at 4 degree overnight.
5. secondary alexa fluor- 1 hr
6. Mounting
(Wash for 5 mins after each step, can be done twice after antibody incubation)
Hope it helps,
Thanks,
Have you tested glutaraldehyde as a fixative instead of PFA?
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