[Histonet] good protocol for ICC in neurons

[Guillermo Palchik]

Dear histonetters, 

I am trying to do some ICC on nuclei of rat primary neurons that have been grown for 2 weeks on coverslips (seeded at 75,000 cells/ml). So far I have followed the protocol that a (long since gone) student in the lab used, but I have been unsuccessful. My nuclei end up looking like raisins....
The protocol that the student used is unlike anything I have seen:
Wash with cold PBS 2X 5 min                  

Rinse briefly with cold methanol 

Fix in methanol at -20C for 20 minutes 

Wash in cold PBS 2 X 10 min 

Block in 5% NGS / 0.05% Triton-X for 6 hours at 4C

Add Primary antibodies diluted in Blocking Solution overnight @ 4C

Wash in cold Wash Solution (2% NGS/0.05% Triton-X) 3 X 10 min 

Add Secondary Antibodies diluted in Blocking Solution for 1.5 hours at RT  in dark

Wash in Wash Solution 3 X 20 min in dark 


Wash in PBS 3 X 10 min in dark 

Add DAPI (1:10,000 in PBS) for 5 minutes in dark

Wash in ddH2O 3 X 10 min in dark

Mount with Fluoro-Gel with Tris

Store @ 4C in dark until dry



First, after a methanol fixation of 20 minutes at -20C the protocol calls for blocking and permeabilizing the cells for 6 hours!

Second, it keeps washing with this washing solution that contains triton.

As I said, I have followed this protocol to the T and I keep getting horrible looking, shriveled cells and nuclei...
The question I have is, does the protocol look okay and maybe it is me, or should I change specific parameters of it?

Does anybody have a good working protocol that could share with me? It would be greatly appreciated...
Thanks
Gil

-- 
Guillermo Palchik
Ph.D. Candidate - Interdisciplinary Program in Neuroscience
Georgetown University Medical Center
Research Building Room W 217
3970 Reservoir Rd. NW, Washington, DC 20007
Lab: 202-687-7825