Cell line: N2102EP & HEK293 Flip-In Trex
Clean the coverslips (1H):
- Wash with concentrated HCl
- Wash with water 10X
- Wash with 70% Ethanol 2X
- Dry in hood
Grow cells on cover slip:
- Grow cells on coverslips 10 - -12 mm diameter for one or two days in 24 well plate:
50% till 70% confluency (two replicates)
Also two wells/coverslips with HEK293T cells..
Fix cells:
- Add formalin 37% (0.1 Volume) on the top of media and let it stay in RT for 30’
- PBS wash 2X
Permeabilize cells:
- Add Triton X100 0.1% in PBS for 10’
- PBS wash 3X
Block cells:
- Block with PBS + 1% BSA solution for 20-30’ RT
Probe with antibody:
- Dilute ORF1 antibody (4H1 1mg/mL) in PBS+1% BSA Solution 1:100
- Incubate cells with antibody for 1 hr in RT
- PBS wash 3X
- Dilute Secondary Alexa-594 conjugate in PBS+1% BSA Solution 1:250
- Incubate cells with secondary Alexa-594 conjugate
- PBS wash 3X
Mount on Microscope slide:
- Mount coverslips on microscope slide using vectashield
- Store at -20 if want to re-probe it
Find below the results:
HEK293t_ORF1_A
HEK293t_ORF1_B
N2102EP_ORF1_A
N2102EP_ORF1_B