Immunofluorescent Staining

Solutions Required

Stock solutions	How to make
4% PFA in PBS	8.75 mL PBS + 1.25 mL 32% PFA
0.5% Tween-20 in PBS	50 mL PBS + 250 µL Tween-20. Add Tween-20 to 1 mL PBS first to mix easier!
0.1% Tween-20 in PBS	40 mL PBS + 10 mL 0.5% Tween/PBS
25% FBS in PBS (5X)	37.5 mL PBS + 12.5 mL FBS
Blocking Solution	40 mL 0.5% Tween/PBS + 10 mL 25% FBS in PBS

• Blocking Solution: 5% FBS, 0.1% Tween in PBS

Adherent Cell Staining

Expected time: 3 days

• Day 1: Fix cells, permeabilize overnight

• Day 2: Incubate with primary antibody overnight

• Day 3: Incubate with secondary antibody + image

1. Remove media and add cold 4% paraformaldehyde (PFA) (in fume hood)

2. Incubate cells in 4% PFA for 1 hour at 4°C

3. Remove 4% PFA (in fume hood)

4. Wash cells 3 times with cold PBS

• Cells may be left in PBS overnight at 4°C

5. If staining nuclear proteins, change solution and incubate cells in 0.5% PBS/Tween for 1 hour to overnight at 4°C to permeabilize

Note

Overnight is preferred for the permeabilization step

6. Change solution and incubate cells in blocking solution for 1 hr

• Cells may be left in blocking solution overnight at 4°C

7. Change solution and incubate cells in primary antibody (diluted in blocking solution) overnight at 4°C

8. Change solution and wash cells with 0.1% Tween/PBS quickly three times.

9. Incubate cells in 0.1% Tween/PBS for 20 min

• Cells may be left in 0.1% Tween/PBS overnight at 4°C

10. Change solution and incubate cells in secondary antibody for 1 hr (secondary antibody is diluted 1:300-500 in blocking solution)

• You can incubate your cells in secondary antibody overnight at 4°C

11. Change solution and wash cells with 0.1% Tween/PBS quickly three times.

12. If doing a DNA stain, add Hoechst or DAPI diluted in PBS for 10 minutes at room temp, then wash three times with PBS

13. Either store in PBS or remove solution and add vectashield solution. Your cells can stay in the solution at 4°C for a long time before you image them, just make sure to parafilm the sides to the liquid does not evaporate

Note

1. Never remove all of the media from the cells-they will dry and absorb antibody in a non-specific way, thereby skewing your results

2. Unless otherwise noted, everything is performed at room temp

3. Keep your antibodies on ice at all times

4. Generally, 50 µL/96-well for all steps is good (can go higher for PBS wash steps)

5. If mounting, try to avoid bubbles in the final step. Leave some solution still on your slides so the cells do not dry and carefully dispense 2-3 drops of the solution, very carefully putting the coverslip on your slide

6. Be very gentle with neuronal cultures, they are not very adherent

Cell Staining for Flow

Expected time: 1-2 days (only 2 if overnight primary antibody)

1. Dissociate cells (trypsin or DNase/Papain if MNs) and spin down

2. Remove supernatant and incubate cells in 3.7-4% paraformaldehyde (PFA) (in fume hood) for 15 min

   • EU calls for 3.7% PFA

3. Add 1 mL PBS and spin cold at 4°C to pellet cells

4. Aspirate solution and incubate cells in 0.5% Tween/PBS for 15 min to permeabilize

5. Add 1 mL PBS and spin cold at 4°C to pellet cells

6. Aspirate solution and incubate cells in 200 µL primary antibody (diluted in blocking solution) for 1 hr in the rotator at 4°C

Note

Some antibodies (e.g. Ki67) work better overnight in the rotator at 4°C

7. Add 1 mL PBS and spin cold at 4°C to pellet cells

8. Aspirate solution and incubate cells in 200 µL secondary antibody (diluted in blocking solution) for 30 min in the rotator at 4°C in the dark

9. Add 1 mL PBS and spin cold at 4°C to pellet cells

10. If doing a DNA stain, add Hoechst or DAPI diluted in PBS for 10 minutes at room temp, then wash with PBS

Note

All spins are performed at ~500 rcf for 5 min. Our centrifuge follows RCF = 1e-4*[rpm]^2 + 4e-2*[rpm] - 6e1, where 2200 rpm = 512 rcf. It is recommended to perform all spins at 4°C once the cells have been fixed to prevent pellet loss.