Objective
Set up IHC free-floating technique with glabrous rat skin. We’ll see neurofilament and try CASPR to detect nodes.
Materials
see rat skin IHC 3
- Mouse-anti-CASPR
Procedure
- Cut the footpad with cryostat (60um) and put them to a 24-well plate with PBS
- Wash with PBS x2
- Wash with PBS-tritón 0,3% 10minutes in agitation x2
- Block: goat serum 5% – PBS-tritón 0,3% 1hour in agitation
- Primary antibody O/N at 4ºC in agitation: (1+2+3) rabbit-anti-neurofilament 1:2000 + mouse-anti-CASPR 1:1000
- Put the plate about an hour at room temperature
- Wash with PBS-tritón 0,3% 10 minutes in agitation x3
- Prepare and filter secondary antibody. Incubate 2hours and a half in agitation and darkness: (1+2+3) GAR-594 1:500 + GAM-488 1:500
- Wash with PBS-tritón 0,3% 10 minutes in agitation x5
- Wash 1 time with PBS 10minutes
- Put the cuts in slides
- Dehydrate with alcohols (A50 / A70 / A96 / A100) 10 minutes each
- Mount with cytoseal
Results & conclusions
We can’t see as many nerves as the previous IHC. Positive mark for CASPR is weak but we can identify some nodes.
Photos: CASPR marks are in green and neurofilament in red.
We’ll try to see more nodes trying to mark myelin with MBP (we’re waiting for the new antibody).
