Thank you Kim Carnes, UIUC, for the image and protocol for the Ret staining of spermatagonial stem cells.
Depraffinize and hydration
Peroxidase blocking - 30 minutes in 0.06% H2O2 in methanol.
Antigen retrieval - 0.01M Citrate buffer (pH6.0) in microwave (10 minutes, power level 7)
Serum blocking - horse serum (unfiltered normal, diluted1:100) for 30 minutes.
Primary antibody - Neuromics Ret antibody (1:200), overnight at 4º C in a moisture chamber.
Secondary Ab - this was a horse universal biotinylated ab that came in a kit (with the above horse serum) diluted 1:50 with PBS and horse serum (also 1:50) for 60 minutes at room temp.
ABC incubation (Vector ABC Elite kit) - 30 minutes.
Colorization with DAB, counterstain with hematoxylin followed by dehydration.
We use PBS rinses between steps.
Thank you Alex Vila, UCLA, for your image and protocol for the SHANK 1a C-terminus staining of retinol tissue
Species: Mouse C57B6
Fixation: 15 minute 4% paraformaldehyde fixation
Antibody dilution: 1:800 overnight.
Secondary Alexa 568 1:500 for 45 minutes
Tissue preparation: Blocked for 2 hrs at room temp in 10% normal goat serum and
Triton X-100 (0.5%) in 0.1 M Phosphate buffered saline (PBS)
Washed: 3X 0.1 M PBS between primary and secondary step.
Mounted tissue with Paramount.
Confocal: Images are 2048 X 2048, 12 bit, collected with a 40X water objective, 10 stacks 0.55 um per stack