Objective
In the last experiment we can see nodes very well, even better than teasing technique. So, we try to do it with sera. We’ll try with LAC (neurofascin control) and with MASP (contactin control).
Materials
see 20140305 1st rat nerve IHC free-floating
Procedure
- Cut the cryopreserved nerve (saved in the last experiment) in the criostat 60um and put the cuts in a 24-well plate with PBS
- Wash with PBS (x2)
- Wash with PBS-tritón 0,3% 10 minutes in agitation (x2)
- Block: Goat serum (PBS-tritón0,3%+Goat5%) in agitation 1h
- Primary antibody o/n 4ºC in agitation: (1) mouse-anti-CASPR 1:1000 + LAC serum 1:100 (2) mouse-anti-CASPR 1:1000 + MASP serum 1:100
- Put the plate about an hour at room temperature
- Wash with PBS-tritón 0,3% 10minutes in agitation x3
- Prepare and filter secondary antibody, then incubate o/n in darkness and agitation at 4ºC. (1+2) GAH-488 1:500 + GAM-594 1:500
- Put the plate about an hour at room temperature
- Wash with PBS-tritón 0,3% 10minutes in agitation 5 times
- Wash with PBS
- Put the cuts in a slide and let dry
- Dehydrate with alcohols (A50, A70, A96 and A100) 10 minutes each
- Mount with cytoseal
Results & conclusions
We can see nodes marked with CASPR and with serum, but sera also mark axons. So, There are too many positive marks from sera and nodes aren’t clear.
To sum up, it isn’t better than teasing technique for seeing nodes.
