20140212 rat skin IHC 1

Objective

Set up a new technique, rat skin IHC

Materials

• Paraformaldehyde
• Sucrose 30% + azide 15mM / PBS
• 24-well plate
• PBS
• Cryostat (OTC, criomold)

• Goat serum
• Ab(mouse)-MBP(Ab62631)
• Ab(rabbit)-neurofilaments (04-112millipore)
• Neurofascin serum (LAC)
• Contactin serum (HVH1)
• Ab(mouse)-neurofascin(AF3235)
• Ab(mouse)-contactina(105582)
• GAR-594 1:500
• GAH-488 1:500
• GAM-594 1:500
• GAC-488 1:500
• GAM-488 1:500
• EtOH 50º
• EtOH 70º
• EtOH 96º
• EtOH 100º
• DPX

Procedure

1.  Cut non-hairy rat skin and put it in paraformaldehyde in PBS.
2. Put it at 4ºC in agitation ~24h.
3. Change paraformaldehyde for sucrose 30% sucrose + azida 15mM in PBS. Put it again at 4ºC in agitation at least 7 days.
4. 18.02.2014/ Clean tissue.
5. Save 7 in the fridge. Put 2 with OTC on a criomold per each  in the correct orientation and freeze it. Then more OTC in the stage and freeze it together.
6. Prepare a 24-well plate with PBS.
7. Cut the tissue with cryostat (60 µm) and put it in the wells. Approximately 20 cuts per each.
8. Wash with PBS (x2).
9. Block: Goat serum (PBS+Goat) in agitation ~1h.
10. Primary antibody o/n 4ºC in agitation: (1) Neurofilaments 1:2000  (2) Neurofascin serum 1:100 + MBP 1:500 (3) Contactin serum 1:100 + MBP 1:500 (4) MBP 1:500 (5) Ab-neurofascina 1:500 + MBP 1:500 (6) Ab-contactina 1:500 + Neurofilaments 1:2000.
11. 19.02.2014/ Put the plate about 1h at room temperature.
12. Wash with PBS in agitation 3-5 times.
13. Secondary antibody o/n 4ºC in agitation: (1) GAR-594 1:500 (2) GAH-488 1:500 + GAM-594 1:500 (3) GAH-488 1:500 + GAM-594 1:500 (4) GAM-594 1:500 (5) GAC-488 1:500 + GAM-594 1:500 (6) GAM-488 1:500 + GAR-594 1:500.
14. 20.02.2014/ Put the plate about 1h at room temperature.
15. Wash 6 times with PBS in agitation and dark.
16. Put the cuts in slides (1 per condition) in the same direction.
17. Let dry.
18. Dehydrate with alcohols: (1st) EtOH50º (2nd)EtOH70º (3rd)EtOH96º (4th)EtOH100º, 10 minutes each.
19. Mount with DPX.

Results & Conclusions

(1) Rabbit-A-Neurofilament: Quit external layer, too much autofluorescence. Filter Ab (precipitate). Try to permeability. Try Ab-PGP as a positive control.

(2) Mouse-A-MBP: Don’t use mouse-Ab because secondary Ab detects everything, maybe rabbit-Ab. Serum-NF: It’s difficult to find nodes because we cannot see the nerves. Look for Ab-myelin brands in papers.

(3) Serum-CNTN+Mouse-A-MBP: Bad dehydrate. Mount with clothespins.

(4) Mouse-A-MBP: Like MBP in (2), a lot of background.

(5) Chicken-A-NF: marks NF155 and NF186, in serum we only see NF155. Search NF marks in earlier biopsies. We can see a nerve going out; We see a continum of NF, why not nodes? Mouse-A-MBP: background.

(6) Ab-neurofilaments: little fibers nearly touching the skin. We see an interesting central neurofilament. Mouse-A-CNTN: a lot of background; positive marks in epidermal’s entrances; receptors with CNTN?