Immunofluorescent Staining

Solutions Required

Stock solutions	How to make
4% PFA in PBS	Pre-made, found in Olaf OR 3 mL PBS + 1 mL 16%PFA from an ampoule OR 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 at room temperature 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 at room temperature or overnight at 4°C

   • 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 30 min - 1 hr at room temperature or overnight at 4°C (secondary antibody is diluted 1:300-500 in blocking solution)

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

    • If staining with DAPI or Hoechst, this can be added to the secondary antibody mixture.

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). AMB has used as low as 35 µL/96-well with comparable results.

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

General notes:

• Cells are usually stained in U- or V-bottom 96-well plates but they can be stained in any container (e.g. test tubes, Eppendorf tubes, polystyrene round-bottom tubes, cluster tubes).

• Antibody dilutions will require optimization.

• Primary antibodies are generally good at 1:500 dilution, Secondary antibodies ~1:1000, Live/Dead (e.g. Zombie) ~1:1000.

Tip

It is recommended to stain with ice-cold reagents/solutions and at 4°C, since low temperature prevents modulation and internalization of surface antigens which can produce a loss of florescence intensity.

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, at room temperature) for 15 min

   • EU protocol calls for 3.7% PFA, AMB uses this for antibody staining as well.

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 at room temperature to permeabilize

Note

Permeabilized cells do not pellet as well. Be careful not to aspirate the cell pellet when aspirating solution after centrifugation.

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

6. Aspirate solution and incubate cells in 100 µL primary antibody (diluted in blocking solution) for at least 1 hr in the rotator at 4°C

Tip

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

7. Add 1 mL 3% FBS (diluted in PBS) and spin cold at 4°C to pellet cells

   Note

   AMB has found using FBS in wash buffer improves cell yield.

8. Aspirate solution and incubate cells in 100 µL secondary antibody (diluted in blocking solution) for 30 min in the rotator at 4°C in the dark

9. Add 1 mL 3% FBS (diluted in 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 cold Eppendorf 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.