Objectives
From byotinilated IgG of sera we want to test if patients have any reactivity against human nerve. In this particular experiment we’ll do it with LAC (positive control of NFASC), HVH1 (positive control of CNTN and with AR (seems to have Ab against CD9 by IP, not tested).
Material
- 48-well plate
- PBS
- Human nerve
- OCT
- Cryostat
- PBS-Triton 0.3%
- Goat serum 5% – PBS-triton0.3%
- Streptavidina-HRP (for blocking)
- Byotilinated IgG from patients sera:
- LAC
- HVH1
- AR
- Ctrl
- Ab-CASPR (Ab105571)
- GAM-488
- Streptavidina-594
- Alcohols (EtOH50, EtOH70, EtOH96, EtOH100)
- Cytoseal
Plate distribution (well nº)
- Ctrl 1/20
- AR 1/20
- HVH1 1/20
- LAC 1/20
- Ctrl 1/40
- AR 1/40
- HVH1 1/40
- LAC 1/40
- HVH1 1/100
- LAC1/100
Methods
From cryopreserved and freezed (-80ºC) in OCT human nerve.
- Prepare a 48well plate with PBS in each well
- Cut the nerve at 60um of thickness and put 2-3 cuts in each well
- Wash 2 times with PBS
- Wash and permeabilize 2 times with PBS-triton 0.3% 5minutes in gentle agitation
- Block with streptavidina 1/500 in Goat serum 5% in PBS-triton 0.3% 2hours in agitation
- Incubate with byotinilated sera 1/20, 1/40 or 1/100 diluted in goat serum 5% in PBS-triton 0.3% o/n 4ºC in agitation
- Incubate with Ab-CASPR 1/500 1h en agitació
- Wash 2 times with PBS
- Incubate with secondary antibodies (streptavidina-594 3/500 + GAM-488 1/500)
- Wash with PBS-triton 0.3% 2 times
- Wash with PBS 1 time and mount the cuts in slides
- Dehydrate with alcohols (A50, A70, A96, A100) 5 minutes each
- Mount with cytoseal
Results
We can see positive marks for CASPR but not for the byotinilated IgG of patients.
Conclusions
It is posible that the byotinilates IgG were too diluted. We need to test it again.
