Please leave your comments or questions

Immuno fluorescence on adherent cells

Author: Gal Haimovich, updated date: , view: 182, Q&A: 0

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


  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)

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.