Objective
In the last ICC we can not see positive marks for antibody against contactin, so we decide to test the new aliquote, the old aliquote and a serum from positive control HVH1.
Materials
- CNTN1-transfected HEK cells, grown in coverslips and freezed
- CNTN1-transfected HEK cells, grown in coverslips, permeabilized and freezed
- Sera from 1 positive controls (HVH1)
- Antibody against CNTN1 (old and new)
Procedure
- Take one coverslip per condition (6)
- Prepare primary antibody diluted in goat serum. (1+2+3) non-permeabilized cells (4+5+6) permeabilized cells. (1+4) old antibody 1:500 (2+5) new antibody 1:500 (3+6) HVH1 serum 1:40. Add a drop (40µl) of each preparation to the parafilm. Incubate for 1h
- Wash 3 times with PBS
- Prepare secondary antibody: (1+2+4+5) GAM-488 1:500 (3+6) GAH-488 1:500
- Wash 3 times with H2O
- Mount with Vectashield (with DAPI)
Results & conclusions
We can see positive marks for HVH1 serum in bothcoverslips (permeabilized and non-permeabilized).
Antibody against CNTN has positive marks in non-permeabilized cells but not in permeabilized.
So, both antibodies are good and them are no appropiate for ICC with permeabilized cells.
