20140305 rat nerve IHC – free floating 1

Objective

We’ll do the same experiment but with rat nerve instead of skin trying to verify the technique because we know how nerves looks like.

Materials

see rat skin IHC 3

• Rat ciatic nerve

Procedure

1. Remove ciatic rat nerve and put it in paraformaldehyde in PBS
2. Put it at 4ºC in agitation ~24h
3. Cut the external part of the nerve and cut the nerve into 3 pieces
4. Change paraformaldehyde for sucrose 30% sucrose + azida 15mM in PBS. Put it again at 4ºC in agitation until it has the same density (~a week)
5. 10.03.2014/Move one piece of nerve away from sucrose to cut it. The others stay with sucrose
6. Cut the nerve in the criostat 60um and put the cuts in a 24-well plate with PBS
7. Wash with PBS (x2)
8. Wash with PBS-tritón 0,3% (x2)
9. Block: Goat serum (PBS-tritón0,3%+Goat5%) in agitation 2,5h
10. Primary antibody o/n 4ºC in agitation: (1) rabbit-anti-neurofilaments 1:2000  + mouse-anti-CASPR 1:1000
11. Put the plate about an hour at room temperature
12. Wash with PBS-tritón 0,3% 10minutes in agitation x3
13. Prepare and filter secondary antibody, then incubate o/n in darkness and agitation
14. Put the plate about an hour at room temperature
15. Wash with PBS-tritón 0,3% 10minutes in agitation 5times
16. Wash with PBS
17. Put the cuts in a slide and let dry
18. Dehydrate with alcohols (A50, A70, A96 and A100) 5 minutes each
19. Mount with cytoseal

Results & conclusions

We can see positive marks for CASPR.

Mark for neurofilament is only positive when NF is cut but no throughout the tissue, so merge of this 2 marks don’t colocalize.

So, free-floating technique is appropriate to see nodes.

And we’ll try with patient’s sera (LAC and MASP; neurofascin-positive and contactin-positive). We’ll do this to see if is better than teasing technique or not.