Immunofluorescence

Please find attached our sequential single-molecule fluorescence in situ hybridisation and immunofluorescence protocol. 

For any queries please email jeff.lee@bioch.ox.ac.uk. 

Sequential smFISH + Immunofluorescence for adherent cells

Coverslip/Imaging chamber preparation

Round coverslips

1. Clean 12-13 mm round coverslips (#1.5) with 80% EtOH-soaked kimwipe
   a. Store the cleaned coverslips in 100%EtOH
2. Place coverslips in a 24-well plate and completely dry them before seeding cells
   a. Seed 0.5-2 million cells per well for 24 hrs
    

IBIDI 8-well chamber (Cat. 80827)

1. Open a new IBIDI 8-well chamber (µ-slide glass)
   a. Seed 0.5-2 million cells per well for 24 hrs

smFISH

1. Aspirate growth medium, and wash 1x with PBS
2. Fix in 4% PFA (in PBS) for 30 minutes at RT
   1. Rinse 1x → Wash 2x with PBS, 5 min ea.
   2. Cells can be stored at 4˚C in PBS with 2 mM RVC for ~ 2 weeks
3. Permeabilise cells in PBSTx 0.1% for 10 minutes at RT
   1. Rinse 1x → Wash 2x with PBS, 5 min ea.
   2. Wash 1x with 2x SSC for 5 min
4. Pre-hybridise 2x in wash solution (pre-warmed to 37˚C) for 20 min ea. at 37˚C
5. Hybridise in hybridisation solution (pre-warmed to 37˚C) with FISH probes overnight at 37˚C (protect from light from this point forward)
   1. 1:50 of 25 µM CoV-2 probes
   2. 1:100 of 25 µM host RNA probes
6. Rinse 1x with pre-warmed wash solution
7. Wash 2x with wash solution, 30 min ea. at 37˚C
8. Wash 2x with 2X SSC, 5 min ea. at RT
9. Wash 1x with PBS, 5 min
    

Immunofluorescence

1. Block for 30 min at RT
   1. Blocking solution: 1% BSA in PBSTw 0.1% - Treat with RNAsecure(1:40) for 30 min at 60˚C
   2. Add 1:100 200 mM RVC after heat treatment
2. Incubate in primary Ab solution (diluted in blocking solution)
   1. dsRNA, Tubulin - 2 hr at RT
   2. anti-RBPs - overnight at 4˚C
3. Rinse 1x → Wash 3x with PBSTw 0.1%, 10 min ea.
4. Incubate in secondary Ab solution (diluted in blocking solution) for 1 hr at RT. Add counterstains as necessary.
   1. Alexa Fluor secondaries 1:500
   2. DAPI (0.5 µg/ml stock) 1:500
   3. HCS CellMask green (Thermo H32714)) 1:800,000
   4. Phalloidin AF488 (Thermo A12379) 1:5,000
5. Rinse 1x → Wash 3x with PBSTw 0.1%, 10 min ea.
6. Wash 1x with PBS, 5 min
7. Mount using antifade mounting medium
        a. Coverglass: 7-10 µl hard-set vectashield OR 10 µl wet-set vectashield and seal it which nail polish
        b. IBIDI 8-well chamber: 100 µl wet-set vectashield OR 3 drops of IBIDI mounting medium per well

Buffers (All solutions made with nuclease-free water)

• Wash solution (Make fresh): 2x SSC + 10% Formamide
• Hybridisation solution (Makefresh): 2x SSC + 10% Formamide + 10% Dextransulphate (w/v)
• PBSTx: 1x PBS + 0.1% Triton-X 100
• PBSTw: 1xPBS + 0.1% Tween-20
   

Materials