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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
