20150417 Teasing (1-6)

Objectives

To screen new patients’ sera from

Materials

• Acetone
• PBS
• Goat sera 5% in PBS
• Dakocytomaton marker¿?
• mouse-a-CASPR 105571
• Patients’ sera (1, 2, 3, 4, 5, 6, HVH1 and a negative control)
• GAH-594
• GAM-488

Methods

1.  Fix teased fibers with acetone at -20ºC for 10 minutes
2. Wash 3 times with PBS
3. Block with goat 5% in PBS for 30 minutes
4. Incubate with commercial antibody (m-a-CASPR-105571) diluted 1/500 in blocking buffer for an hour
5. Wash 3 times with PBS
6. Incubate for 1hour with sera diluted 1/100
7. Wash 3 times with PBS
8. Incubate with secondary antibodies (GAH-594 + GAM-488) 1/500 for an hour
9. Wash 3 times with H2O
10. Mount with fluoprep

Results

We can see positive marks in all of patients but also in the control. Patient nº4 is more positive in nodes of Ranvier.

Conclusions

We’ll repeat it with more healthy controls.