20140305 rat nerve IHC 7um

Objective

Is a positive control for 20140305 rat nerve IHC free floating

Materials

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

• Goat serum
• Ab(rabbit)-neurofilaments (04-112millipore)
• Ab(mouse)-CASPR
• GAR-594 1:500
• GAM-488 1:500
• EtOH 50º
• EtOH 70º
• EtOH 96º
• EtOH 100º
• Cytoseal

Procedure

1. Freeze fixed and cryoprotected nerve with OCT
2. Cut it at 7um and put it in slides. Some slide are stored at -20ºC
3. Encircle cuts with DakoCytomation
4. Wash with PBS 2times
5. Block with PBS-serum Goat 5% 1hour
6. Incubate primary antibody o/n at 4ºC: mouse-anti-CASPR 1:1000 + rabbit-anti-Neurofilament 1:2000
7. Put the slide at room temperature
8. Wash with PBS 3-5 times
9. Incubate secondary antibody 1hour in darkness: GAR-594 1:500 + GAM-488 1:500
10. Wash 6 times with PBS 5 minutes each in darkness
11. Mount with fluoroprep and let dry

Results & conclusions

There are positive marks for CASPR and Neurofilament. It’s possible to see nodes (CASPR positive) that maybe colocalize with neurofilaments, but like in the last experiment neurofilament it isn’t positive throughout the cut.