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Immuno fluorescence on adherent cells

Author: Gal Haimovich, updated date: , view: 38, Q&A: 0
Tags: IMMUNOFLUORESCENCE, IF and staining

Materials and Reagents

  1. PBSM (1x PBS + 0.5 mM MgCl2)

  2. PFA

  3. Glycine

  4. Triton-X-100

  5. BSA

  6. primary antobody

  7. PBT (1x PBS+ 1% Triton)

  8. DAPI

  9. Coverslips

  10. Glass slides

  11. Prolong Gold

  12. Nail polish



Procedure

  1. Wash with PBSM (=PBS + 5mM MgCl2) (3 quick rinses)

  2. Fix cells 10 min RT in 4% Paraformaldehyde in PBSM

  3. Wash with 0.1M Glycine/PBSM 10 min @ RT (quenching)

  4. Wash with PBSM 2 x 10 min @ RT (cells can be left overnight @ 4°C at this point).

  5. Permeabilize cells 5 min with PBS supplemented w/ 0.2% triton x100. (0.2% PBT)

  6. Blocking: incubate for 30' in 0.1% PBT with 3% BSA.

  7. Place coverslip over 50μl of primary Ab diluted in 0.1% PBT with 3% BSA in a humid chamber for 2h at RT.

  8. Wash 3x5' with PBT 0.1%.

  9. Place coverslip over 50μl of second Ab  diluted in 0.1% PBT with 3% BSA in a humid chamber for 20-30 min at RT.

  10. Wash 3x5' with PBT 0.1%.

  11. Wash 5min in PBS.

  12. Mount coverslip onto a slide using mounting medium (prolong gold+DAPI).


Note: Always have negative control of the secondary antibody (without the primary antibody)


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