OBJECTIVE: To test myelinated fibers IHC in foot pads from a rat. We will perform 2 different conditions for MBP + PGP and for Caspr + MBP.
IMMUNOFLUORESCENCE (in humid chamber):
1st day:
- Air-dry slides for 30 min and thaw one or two blocking aliquots.
- Outline the sample boundaries with DAKO-PEN.
- Block 30min at room temperature with 10% BSA in PBS.
- Preparation of the primary antibody
a. 1%BSA (1ml 10%BSA+9ml PBS)
b. 1%BSA + 0.3%TritonX (1ml 1%BSA + 3ul TritonX) (vortex).
c. For PGP + MBP: PGP 9.5 (polyclonal rabbit-to-human) (1:200) diluted in 1%BSA + 0.3% TritonX. MBP (polyclonal mouse-to-human) (1:200) diluted in 1%BSA + 0.3%TritonX. d. For caspr1 + MBP: Caspr ab133634 (1:200) + MBP (polyclonal mouse-to-human) (1:200) diluted in 1%BSA + 0.3%TritonX. - Remove the blocking solution (tapping on paper, no washing).
- Put the primary antibody on the sections (approx. 40uL in each).
- Incubate at room temperature overnight.
2nd day:
- Wash the slides in PBS (x3) for 5 min.
- Preparation of secondary antibodies – Dilutions in 1% BSA in PBS:
a. PGP + MBP – Cy3 conjugate 1:50 + GAM488 A11001 1:100
b. PGP + MBP: GAR488 260214 1:100 + GAM594 A11005 1:100
c. Caspr1 + + MBP: GAR488 260214 1:100 + GAM594 A11005 1:100
d. Caspr1 + MBP: GAR594 A11037 1:100 + GAM488 A1001 1:100
- Put the secondary antibody on the slide (40uL) for 2h at room temperature and in the dark.
- Wash with PBS x3.
- Cover the slides with Mounting media: Vectashield-DAPI.
- Cover the edges with nail polish or Vitro-Cloud (to prevent the preparations from drying out).
- Store at 4ºC in the dark until reading. Once analyzed, they are frozen at -20ºC, where they can last for years and can be re-analyzed in the future if necessary.
RESULTS: Myelinated fibers staining with MBP looks well, maybe more background with GAM488. I cannot see Caspr1 staining, we will try to concentrate the primary antibody Caspr1 to 1:50 and different stainings for the nodes and paranodes: Neurofascin and PanNachannel.
PGP 
MBP 
PGP 
MBP
