Protocol: Immunofluorescence / confocal microscopy

B or T cells in suspension, adherent cells on chambered coverglass or chamberslides, cryostat sections of unfixed, OCT embedded tissue:

1. wash cells 1x cold RPMI (no wash for cryostat sections).

2. Fix 20 min 4% paraformaldehyde in 0.1M phosphate buffer pH 7.4, 0.03M sucrose on ice. **

3. wash 2x PBS/1%BSA. From now on everything can be at room temp. or on ice.

4. Permeabilize 5 min RT in 0.2% saponin, PBS, 0.03M sucrose, 1% BSA.

5. wash 1x PBS/BSA.

6. Block 15 min 5% normal goat serum (NGS) in PBS/BSA.

7. wash 1x PBS/BSA.

8. 1 diluted in PBS/BSA 60 min RT; 100 l per tube or section.

9. wash 3x PBS/BSA.

10. Block 15 min 5% NGS in PBS/BSA.

11. wash 1x PBS/BSA.

12. 2 diluted in PBS/BSA 30 min RT; 100 l per tube or section.

13. wash 3x PBS/BSA; (wash 1x in PBS/BSA then 2 x 10 min in Molecular Probes SlowFade Light buffer if using Slow Fade Light S-7461 to coversip); pellet cells and put up in two drops of Molecular Probes Slowfade Light antifade medium. Pipet about 15 m l on slide and coverslip. For chambered coverglass just put two-three drops in each chamber after wash.

 

**This is a "light" fix due to the sensitivity of the antigen(s). Cells should be imaged as soon as possible.

 

 

Another fix for cryostat sections:

(Unfixed tissue embedded in OCT and frozen)

1. Fix slides with sections (using VWR Superfrost Plus slides) in -20 acetone for 10 min.

2. wash 3x PBS/0.5% BSA.

3. Block and label as above #6-13 except use 0.5% BSA in wash with PBS. Use ProLong antifade to coverslip.

 

Notes:

When looking at B cells (they are relatively fragile) I have tried to support the coverslip with EM grids on two sides, 12 m m diameter latex beads from Sigma (LB-120) mixed with the final cell suspension in antifade medium, and have tried #1 coverslips instead of #1.5. Using #1 coverslips has given me the fewest smashed cells. I now use an inverted scope and pipet the cells onto coverslip chamber slides (Lab-Tek #136439) in antifade medium so no worries about flattened, smashed cells.

smashed by coverglass

looking pretty good

To look at cytoskeleton I have fixed adherant cells on chambered coverslip slides and cryostat sections with ice cold methanol:acetone 1:1 for 10 minutes, then in ice cold ethanol:acetic acid 95:5 for 10 minutes, then wash in saline. Block and label as above steps 6-13.

keratin in cultured human mesangial cell

keratin in cryostat section kidney proximal tubule

Vectashield (Vector Laboratories H-1000 and H-1200 with DAPI) antifade medium gives better antifade protection than SlowFade Light but is hard on the cells; I see lots of membrane blebbing on both fixed B cells in suspension and on fixed adherent melanoma cell lines. Sometimes the problem is worse than others; sometimes with Slowfade Light I see blebbing. On tissue sections which can be allowed to air dry, Molecular Probes ProLong Antifade Kit (P-7481) is the best.

 

cell with blebs in Vectashield

smashed cell with blebs in Slowfade Light

 

1% Triton-X completely destroyed fixed B cells lines in my hands. O.5% saponin as above does a good job of permeabilizing most of the cells and leaving them structurally intact.

Using 50mM NH4Cl in PBS or 0.15 M glycine in PBS to "quench" cells after paraformaldehyde fixation had absolutely no effect. I do not use either anymore.

Coating slides with poly-L-lysine (or using Sigma Poly-Prep slides P-0425) to make these cells in suspension stick to the slide did not help - they stuck maybe a bit but the coated slides picked up the secondary fluorescent tag somehow and were noisy. If you need a coated slide for your work use VWR Superfrost Plus #48311-703; sections stick throughout the labeling process and the slide does not give a noisy background.

Cells are fragile; handle gently even when fixed; do not vortex them. I routinely run cells up in Falcon #2054 tissue culture tubes, not microfuge tubes, and use low speed spins to pellet - just enough to get the cells down in 3-5 min. This way the cell "pellet" is always soft and easy to resuspend with gentle swirling. On chambered coverglass I make sure that the addition and removal of solutions is done gently in the corner of the chamber so no cells are hit with a stream of any solution.

Tips from Susan Anderson 543-9705