IF Staining Protocols

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Please note: This is a general protocol that provides a good start for testing a new sample, target, or antibody. The protocol has been developed by Proteintech’s R&D Team. It may, however, require further adaptation for each specific experiment.

Fixation and Permeabilization
  1. Aspirate medium, then briefly wash cells seeded on clean glass coverslips with 1X PBS.

  2. Fix the cells for 10–20 min. Rinse coverslip with 1X PBS 3 times for 3 min each.

  3. Treat coverslip with permeabilization buffer (e.g. TritonX-100, 0.1 to 1% in PBS) for 10–20 min at room temperature. Rinse coverslip 3 times with 1X PBS for 3 min each.


4. Prepare blocking solution. Incubate the cells with blocking solution at room temperature for 1 h.

Primary & Secondary Antibody Incubation
  1. Aspirate blocking solution, apply primary antibody diluted in antibody dilution buffer. At the same time, set up a negative control without applying the primary antibody. Incubate for 1 to 2 h at RT or overnight (ON) at 4°C in the dark.

  2. Wash coverslip with 1X PBS 3 times for 3 min each.

  3. Apply corresponding fluorochrome-labeled secondary antibody diluted in antibody dilution buffer and incubate for 1 h at room temperature in a moist environment in the dark.

  4. Wash coverslip with 1X PBS 3 times for 3 min each.

Mounting and Visualization
  1. Mount coverslip. Optional counterstain with DAPI for nuclei detection.

  2. Examine slides at a microscope.


Solutions Needed
1X PBS 1000 ml Antibody dilution buffer 100 ml
10 mM Na₂HPO₄ 1.42 g 1% BSA 1g
1.8 mM KH₂PO₄ 0.24 g Add 1X PBS to 100 ml -
137 mM NaCl 8 g - -
2.7 mM KCl 0.2 g - -
Adjust pH to 7.4; Add ddH₂O to 1000 ml - -