ICC/Immunofluorescence Protocols for Cell Climbing Slices

IHC Technical Resources

Protocols, optimization tips,
troubleshooting guides,
and more for IHC.

Troubleshooting guides

Troubleshooting guides

Download troubleshooting
handbooks for IHC, Western
blot and ELISA for FREE.

Summary workflow chart for ICC/Immunofluorescence protocol:

ICC/IF Workflow with Applicable Boster’s Reagents

  1. Cell Climbing Slice Preparation

    • Place settled coverslip in culture bottle or perforated plate
    • Take out coverslip after cell growth has reached 60%
    • Wash the coverslip 3X with PBS to remove culture medium
    • Immerse the coverslip (cells face up) into cold acetone or 4% paraformaldehyde or neutral formalin for 10 to 20 min (Close the lid to prevent evaporation)
    • Wash the coverslip 3X with PBS
    • Put the coverslip on filter paper (cells face up)
    • Remove the liquid on the coverslip and allow it to dry for 8-10 hrs
    • To thaw the slice, wash with neutral PBS at room temperature for 10-15 min (The cell climbing slice can be stored in gelatin at -20°C for one week.)

    Note: This fixation procedure using paraformaldehyde and formalin fixatives may cause autofluorescence in the green spectrum. In this case, you may try fluorophores in the (i) red range or (ii) infrared range if you have an infrared detection system.

  2. Inactivation

    • Mix H2O2 with distilled water (v/v: 1:50)
    • Immerse frozen section or cell climbing slice into the diluted H2O2 at room temperature for 10 min
    • Wash the section 3X distilled water (1 min each)
  3. Antigen Retrieval (Proteolytic Induced Epitope Retrieval: PIER)

    • Dry the cell slices with filter paper
    • Add compound digestion solution (e.g. Trypsin solution or other enzymatic antigen retrieval solution) to the slices (We recommend the addition of 0.1% Triton to the samples before the digestion. This reduces surface tension and allows reagents to easily cover the entire sample.)
    • Incubate the slices at room temperature for 10 min
    • Wash with 3X PBS (10 min each)
  4. Blocking

    • Add 5% BSA blocking solution or normal goat serum to the PIER treated samples
    • Incubate the samples at 37°C for 30 min
    • Shake off extra liquid and dry the samples with filter paper (No washing required)
  5. Primary Antibody Incubation

    • Dilute primary antibody with antibody diluent to the concentration recommended by the antibody manufacturer
    • Add the diluted antibody (Recommended concentration: 0.4 µg to 2 µg) to the samples and incubate at 4°C overnight
    • Wash the samples 3X with PBS (15 min each)
  6. Secondary Antibody Incubation

    • Dilute biotinylated secondary antibody with antibody diluent to the concentration recommended by the antibody manufacturer
    • Add the diluted antibody to the samples and incubate at 37°C for 30 min
    • Wash the samples 3X with PBS (8 min each)
  7. Staining

    • Add Strept-Avidin Biotin Complex – Fluorescence Iso-Thio-Cyanate (SABC-FITC) or Strept-Avidin Biotin Complex – Cyanine-3 (SABC-Cy3) reagents to the samples
    • Incubate the samples at 37°C for 30 min (Avoid light)
    • Wash the samples 2X with PBS (Total 2 hrs)
    • Seal the slices with water soluble sealing reagent
    • Monitor the staining intensity under a fluorescence microscope
    • Counterstain by adding DAPI staining solution to the sample
    • Check again the staining intensity under a fluorescence microscope
    • For slide storage without significant decay in fluorescence signal, add 20 µL of anti-fade solution to the sample followed by a cover glass (Avoid bubbles)