Samples: 153
PROTOCOL:
- Fixation: in acetone 10 min at RT
- Wash 2 times with PBS (5 min) with agitation
- Use the marker(DakoPen) to highlight the location of the sample without exerting excessive pressure.
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 0.3% Triton in Goat Serum 5% blockade solution. Incubate 1 hour at RT in a humid chamber .
- Dilute the serum samples 1/100 in 5% goat –> add 200 mcL /sample of diluted serum and incubate 1 h at TA in the humid chamber.
- Dilute commercial primary antibody ANO2 (TA316534 OriGene) diluted 1/100 with goat serum 5% –> Add 200 mcL /sample of the commercial diluted antibody and incubate 1 h at TA in the humid chamber.
- Wash 2 times with PBS (5 min) with agitation
- Secondary antibodies: dilute GAR488 and GAH 594 1/1000 with 5% goat serum –> incubate 1 h at TA in the humid chamber.
- Wash 2 times with PBS (5 min) with agitation
- Mount the preparation with Fluoromount
RESULTS: Inespecific staining, maybe because the teasing is permeabilized. We will have to perform it only with the secondary antibody and do the teasing in healthy controls.
