Objective
Set up IHC free-floating technique with glabrous rat skin. We’ll see neurofilament and MBP to detect myelin fibers.
Materials
see rat skin IHC 2
- Cytoseal
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% 1hous in agitation
- Primary antibody O/N at 4ºC in agitation: (1+2+3) rabbit-anti-neurofilament 1:2000 + mouse-anti-MBP 1:500
- 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 O/N at 4ºC in agitation and darkness: (1+2+3) GAR-594 1:500 + GAM-488 1:500
- Put the plate about an hour at room temperature
- 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)
- Mount with cytoseal
Results & conclusions
- Ab-MBP: It doesn’t work in this experiment.
- The only change with the last experiment is the mounting medium cytoseal instead of DPX, which is for fluorescence. So, we can see neurofilaments better. Thus, we’ll do one footpad with neurofilament and CASPR, trying to localize nodes.
Figure 1: General photo
Figure 2: Epidermal holes
Figure 3: A thick nerve that should have myelin
Figure 4: In green wee have to see MBP but in these photo as an example of all the cuts we only see background, so our conclusion is that we have to change our commercial mouse-anti-MBP.
Figure 5: Merge figure 3 with figure 4. In positive neurofilament is not positive MBP.
Figure 6: Final ramification of nerve
