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Research Technologies Branch

Biological Imaging Section

Protocols: Generalized Protocol for Immunolabeling


  1. If single cells are to be stained, fix in 4% paraformaldhyde (PFA) for 15 to 20 minutes. This should be electron microscopy-grade ultra pure paraformaldhyde, made up in either Phosphate Buffered Saline (PBS) or in cell culture medium. For tissue sections or whole pieces of tissue, longer fixation is required.
  2. Wash 3 x 5 minutes in PBS.
  3. Permeabilize cells in -20°C methanol for 15 minutes in a freezer. An alternate to this can be 0.1% Triton X100 for 15 minutes. Other alternates include Saponin or Tween. Note: This step is not necessary if cells were fixed with methanol or acetone.
  4. Wash 3 x 5 minutes in PBS.
  5. Add primary antibody (made up in PBS with 1% BSA). Allow this to incubate for 1 hour at room temperature in a humid chamber. If you are working with tissue sections, longer times may be required.
  6. Wash 3 x 5 minutes in PBS 1% BSA.
  7. Apply secondary antibody in PBS BSA; allow to sit for 30 minutes.
  8. Wash 3 x 5 minutes in PBS.

    If you are going to dual label, continue below; if not, skip to step 13.
  9. Apply second primary antibody prepared in PBS BSA. Allow primary antibody to react for 1 hour.
  10. Wash 3 x 5 minutes with PBS BSA.
  11. Apply second secondary antibody prepared in PBS BSA; allow to stand for 30 minutes.
  12. Wash 3 x 5 minutes in PBS.
  13. If you are going to stain nuclei, apply DAPI or Hoescht at a concentration of 1ug/ml in dH2O, and allow to stain for 15 minutes. If you are staining for organelle DNA, or other small structures, you can try the DAPI at a concentration of 5 ug/ml.
  14. Apply coverslipping medium. This can be Vectashield (Vector Labs) Pro-Long (Molecular Probes), Fluoroguard, or Mowiol with PPD (mixed before use). Use as little mounting medium as possible to keep the preparations thin. 15ul is enough for a standard 22 x 22 mm coverslip, 9ul for a 13 mm round. If your sample is thick (i.e., a tissue slice), you will require additional mounting medium.

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Last Updated April 23, 2007