Protocols_STORM Cell Fixation and Staining Protocol - BackmanLab/BackmanLab.github.io GitHub Wiki

STORM Cell Fixation and Staining Protocol

All solutions used must be made fresh (immediately before fixation):

Fixative Solution: 3% Paraformaldehyde and 0.1% Glutaraldehyde ​in PBS

Quenching Solution: 0.1% sodium borohydride in PBS

Blocking Buffer: 0.2% Triton X-100 and 3% BSA in PBS (may vary based on cell line)

Washing Buffer: 0.1% Triton X-100 and 0.2% BSA in PBS

  1. Make blocking buffer.

    a. Weigh out BSA and add to centrifuge tube.

    b. Tilt tube so that BSA crystals are spread out, then add PBS (to prevent formation of crystal clumps that will not dissolve).

    c. Leave tube at 4°C until the all crystals are dissolved. DO NOT shake tube or vortex -- this will cause bubble formation which will attract proteins to the surface and prevent BSA from dissolving completely.

    d. Once completely dissolved, add Triton X-100. If crystals are not completely dissolved, use a pipette to mix solution slowly without forming bubbles.

  2. Make washing buffer (follow same steps as blocking buffer).

  3. Make fixative solution.

    a. Add PBS to centrifuge tube.

    b. Add PFA to centrifuge tube.

    c. Add GA to centrifuge and mix.

  4. Take live cells from incubator and wash quickly with PBS.

  5. Fix the cells with fixative solution for 10 minutes. Do all steps from this point forward on a shaker.

    a. While the cells are being fixed, weigh out sodium borohydride for quenching solution and add to centrifuge tube.

  6. Wash the cells with PBS once for 5 minutes.

  7. Add PBS to sodium borohydride in centrifuge tube and mix.

  8. Quench the cells with quenching solution for 7 minutes.

  9. Wash the cells with PBS three times for 5 minutes.

  10. Permeabilize and block the cells with blocking buffer for 20 minutes.

  11. Add primary antibody in blocking buffer (1:2000 or 2.5 µg/mL) and leave rocking for 1-2 hours at room temperature.

  12. Wash the cells with washing buffer three times for 5 minutes.

  13. Add secondary antibody in blocking buffer (1:4000 or 2.5 µg/mL) and leave rocking for 40 minutes at room temperature.

  14. Wash the cells with PBS twice for 5 minutes.

  15. Store cells in PBS at 4°C until ready to image. Wrap the dishes with parafilm so that the dishes do not dry out.

Troubleshooting

  • If there are very few cells when imaging and there are lines on the dish:

    • Problem -- you are pipetting solution directly into the cells and causing them to detach from the dish

    • Solution -- always pipette solution onto the walls of the dish and never directly onto the cells

  • If there are very few cells when imaging without the appearance of any lines:

    • Problem -- not enough cells were on the dish when you began fixing and staining

    • Solution -- make sure to plate more cells onto the dish OR wait one more day to let the cells grow

  • If the cells look stressed (i.e. if there is very little cytoplasm or the nuclei are not clearly visible):

    • Problem -- you are leaving the cells dry for too long when you are switching out solutions

    • Solution -- pipette solutions in and out of the dish quickly and make sure the cells are always submerged in liquid

  • If the cells look hypo- or hypertonic:

    • Problem -- the fixative solution is not properly pH or ion balanced

    • Solution -- make sure the PFA is less then 2 months old and the GA is less than 2 weeks old prior to making fixative, then make sure to make the fixative fresh immediately before fixing cells

  • If there are worm-like structures or blobs that do not look like cells on the dish:

    • Problem -- either the cells or one of your solutions are contaminated

    • Solution -- make sure your cells do not have contaminants prior to fixation (by checking visually under a microscope) and make sure none of your solutions (specifically your PBS source bottle) have bacterial or mold contaminants floating

Solution and Buffer Calculations

For one 4-well slide (750 µL of solution per well):

  • Fixative Solution (3.5 mL)

    • 2.70375 mL PBS

    • 656.25 µL 16% PFA

    • 14 µL 25% GA

  • Quenching Solution (10 mL)

    • 0.01 g NaBH4

    • 10 mL PBS

  • Blocking Buffer (10 mL)

    • 0.3 g BSA

    • 9.8 mL PBS

    • 200 µL 10% Triton X-100

  • Washing Buffer (10 mL)

    • 0.02 g BSA

    • 9.9 mL PBS

    • 100 µL 10% Triton X-100

For one 35 mm dish (1 mL of solution per dish):

  • Fixative Solution (1.5 mL)

    • 1.71275 mL PBS

    • 281.25 µL 16% PFA

    • 6 µL 25% GA

  • Quenching Solution (10 mL)

    • 0.01 g NaBH4

    • 10 mL PBS

  • Blocking Buffer (4 mL)

    • 0.12 g BSA

    • 3.92 mL PBS

    • 80 µL 10% Triton X-100

  • Washing Buffer (4 mL)

    • 0.008 g BSA

    • 3.96 mL PBS

    • 40 µL 10% Triton X-100

Antibody Dilutions

Primary Antibodies (to get final concentration in blocking buffer of 2.5 µg/mL):

  • Anti-RNA polymerase II CTD repeat YSPTSPS phospho S2

    • ab193468 = 3.26 µL for 1 mL blocking buffer

  • Anti-Histone H2B

    • ab52599 = 12.20 µL for 1 mL blocking buffer

  • Anti-Histone H3 tri methyl K27

    • ab192985 = 2.39 µL for 1 mL blocking buffer

  • Anti-Histone H3 tri methyl K9

    • ab176916 = 1.94 µL for 1 mL blocking buffer

  • Anti-Histone H3 tri methyl K4

    • ab213224 = 5.10 µL for 1 mL blocking buffer

    • ab12209 = 2.50 µL for 1 mL blocking buffer

  • Anti-Histone H1.2

    • ab181973 = 0.77 µL for 1 mL blocking buffer

Secondary Antibodies (to get final concentration in blocking buffer of 2.5 µg/mL):

  • Alexa 647 = 1.25 µL for 1 mL blocking buffer

  • Alexa 568 = 1.25 µL for 1 mL blocking buffer

  • Alexa 546 = 1.25 µL for 1 mL blocking buffer

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