Intracellular Staining Protocol: Two Step Fix/Perm

Date Published: July 18, 2023

Two Step Fixation and Permeabilization

Materials: Maintain Reagents and buffers at 4°C.

  1. Fluorochrome-conjugated primary antibody
  2. Fluorochrome-conjugated isotype control antibody
  3. Fixation Buffer, ADI-950-011
  4. Permeabilization Buffer, ADI-950-012
  5. PBS (Phosphate Buffered Saline), OmniPur 6505
  6. FBS (Fetal Bovine Serum), Gibco 10082
  7. 10 x 75mm Tubes, VWR 60818-281

Procedure:

  1. Harvest N x 106 cells and wash twice in 20 ml of PBS followed by centrifugation at 400 x g for 7 minutes (N=number of samples to be analyzed +1 for each isotype control).
  2. Re-suspend cells in N x 1 ml of Fixation Buffer.
  3. Mix well by inverting and incubate for 30 minutes at 4°C.
  4. Mix well by inverting and centrifuge at 400 x g for 7 minutes.
  5. Decant supernatant and re-suspend cells with N x 1 ml of Permeabilization Buffer.
  6. Mix well by inverting and incubate for 10 minutes at 4°C.
  7. Centrifuge at 400 x g for 7 minutes.
  8. Decant supernatant and re-suspend pellet in N x 50 µl of Permeabilization Buffer.
  9. Gently mix by pipette and aliquot the cell suspension in 50 µl aliquots.
  10. Add the appropriate amount of antibody, to the cell aliquots, to achieve the target working concentration.
  11. Gently mix by pipette and incubate for 30 minutes in the dark at 4°C.
  12. Add 1 ml of Permeabilization Buffer.
  13. Gently mix by pipette and incubate for 5 minutes at 4°C.
  14. Centrifuge at 400 x g for 7 minutes.
  15. Add 1ml of Permeabilization Buffer.
  16. Gently mix by pipette and incubate for 5 minutes at 4°C.
  17. Centrifuge at 400 x g for 7 minutes.
  18. Decant supernatant and wash in 1 ml of PBS.
  19. Gently mix by pipette and centrifuge at 400 x g for 7 minutes.
  20. Decant supernatant and wash in 1 ml of PBS.
  21. Gently mix by pipette and centrifuge at 400 x g for 7 minutes.
  22. Decant supernatant and gently re-suspend cell pellet by pipette with 500 µl of PBS containing 1% FBS for evaluation by flow cytometer.

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