- Fixation: in acetone -20ºC for 10 min at RT
- Wash 2 times with PBS (5 min) with agitation
- Use the marker (DakoPen) to highlight the location of the sample.
Indicate the sample number and the date in pencil on the frosted glass and place the samples in the humid chamber. - Blocking: cover the entire sample with Goat Serum 5% with triton 0.1% . Incubate 1 hour at RT in a humid chamber .
- Dilute the serum samples 1/100 in Goat Serum 5% with triton 0.1% –> add diluted serum and incubate 1 h at TA in the humid chamber.
- Wash 2 times with PBS (5 min) with agitation
- Dilute commercial primary antibody Chicken anti-human / rat / mouse-NF (AF3235 R & D System) * 1/500 with 5% with triton 0.1% –> Add the commercial diluted antibody and incubate OVER NIGHT AT 4ºc
- Wash 2 times with PBS (5 min) with agitation
- Secondary antibodies: dilute GAC488 and GAH 594 1/1000 with 5% with triton 0.1% –> incubate 1 h at TA in the humid chamber.
- Wash 2 times with PBS (5 min) with agitation
- Mount with Fluoromount
SAMPLES: see table for samples and results
NEGATIVE CONTROL WITH Caspr1 commercial Ab | negative, I dont see Caspr1 commercial Ab |
---|---|
Regine | Positive: paranodes |
Vieira | Positive: paranodes |
Piffort | Positive: paranodes |
MRVM CNTN1 ABSORBED 1:800 | Positive: paranodes |
MRVM Caspr1 ABSORBED 1:800 | Positive: paranodes |
MRVM CNTN1/Caspr1 ABSORBED 1:800 | NEGATIVE |
MRVM HEK ABSORBED 1:800 | Positive: paranodes |
17595 CNTN1 ABSORBED 1:800 | NEGATIVE (REPEAT) |
17595 Caspr1 ABSORBED 1:800 | NEGATIVE (REPEAT) |
17595 CNTN1/Caspr1 ABSORBED 1:800 | NEGATIVE |
17595 HEK ABSORBED 1:800 | Positive: paranodes |
VIEIRA CNTN1 ABSORBED 1:1000 | Positive: paranodes |
VIEIRA Caspr1 ABSORBED 1:1000 | Positive: paranodes |
VIEIRA CNTN1/Caspr1 ABSORBED 1:1000 | NEGATIVE |
VIEIRA HEK ABSORBED 1:1000 | Positive: paranodes |