I am doing Immuno on cultured hippocampal neurons on coverslips. I found that most of my neurons are dead after doing the immuno.
When I use DAPI, I see many neurons, but once I change to fluoroscence, I find most of my neurons dead. Kindly suggest, what can I do to solve this problem?
I am sharing the protocol that I use below:
1) Remove coverslips and leave them at RT for 10 min (on the culture media).
2) Rinse them once with fresh PBS.
3) Fix the coverslips with 500 µL of 4% PFA one ice for 10 min.
4) Wash them 3 times with fresh PBS for 5min each (3 x 5’).
5) Add 1 ml of blocking solution to the coverslip and incubate them in 37ºC for 30 min. In the meantime, prepare the primary and secondary antibody solution.
6) Add 1 ml of primary antibody solution to the coverslips and incubate them at 37ºC for 45 min.
7) Wash the coverslip 3 times with fresh 1X PBS (3 x 5’).
8) Add 1 ml of secondary antibody solution to all coverslips and incubate them at 37°C for 45 min.
9) Remove the antibody solution and wash the coverslip 3 times with fresh PBS and 5 min each in dark (3 x 5’).
10) Leave the coverslips in water in PBS; place a drop of Prolong Diamond at the center of the slide; mount 1 coverslip/slide and place them gently on the prolong Diamond inverted, so that the cells face the mounting media.
11) Leave the slides for 24 h at RT, in dark, for curing the mounting media (coverslip side facing up).