免疫荧光操作（共聚焦显微镜）-Immunofluorescence Protocol for Confocal Microscopy

互联网2013-12-26

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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

~undefinedThis_is_a_~4light~4_fix_due_to_the_sensitivity_of_the_antigen~As~B._Cells_should_be_imaged_as_soon_as_possible.~K~Hp~M~Kp~M~K~Hp~M~Kp~M~2~1~0~K~Hp~M~Kp~M~K~Hp~M~Kp~M~2~1~0Another_fix_for_cryostat_sections~I~K~Hp~M~Kp~M~K~Hp~M~Kp~M~2~1~0~AUnfixed_tissue_embedded_in_OCT_and_frozen~B~K~Hp~M~Kp~M~K~Hp~M~Kp~M~2~1~01._Fix_slides_with_sections_~Ausing_VWR_Superfrost_Plus_slides~B_in_~F20_° 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.

<center> <table> <tbody> <tr> <td> <p> </p> </td> <td> <p> </p> </td> </tr> <tr> <td> <p> smashed by coverglass</p> </td> <td> <p> looking pretty good</p> </td> </tr> </tbody> </table></center>

¨ 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.

<center> <table> <tbody> <tr> <td> <p> </p> </td> <td> <p> </p> </td> </tr> <tr> <td> <p> keratin in cultured human mesangial cell</p> </td> <td> <p> keratin in cryostat section kidney proximal tubule</p> </td> </tr> </tbody> </table></center>

¨ 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.

<center> <table> <tbody> <tr> <td> <p> </p> </td> <td> <p> </p> </td> </tr> <tr> <td> <p> cell with blebs in Vectashield</p> </td> <td> <p> smashed cell with blebs in Slowfade Light</p> </td> </tr> </tbody> </table></center>

¨ 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 NH 4 Cl 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