IF Protocol Hub

IF Protocol for Cultured Cells - Step-by-Step Procedure

Detailed immunofluorescence protocol specifically optimized for cultured cells. Ready-to-use procedure with cell-specific considerations.

Created: 12/13/2025Updated: 12/13/2025By: IF Protocol Hub

IF Protocol for Cultured Cells

This protocol is specifically optimized for immunofluorescence staining of cultured cells. For general IF procedures, see the General IF Protocol.

When to Use This Protocol

Use this protocol when working with:

  • Adherent cell lines
  • Primary cultured cells
  • Cells grown on coverslips or chamber slides
  • Suspension cells (with modifications)

Pre-Experiment Preparation

Reagents & Materials

Cell Culture Materials

  • Coverslips: Sterile glass coverslips (12-18 mm diameter, #1.5 thickness)
  • Coating solution: Poly-L-lysine, collagen, or fibronectin (if needed)
  • Cell culture medium: Appropriate for your cell type
  • PBS (sterile): For washing cells

IF Staining Reagents

  • Fixative: 4% PFA in PBS (or methanol/acetone)
  • Permeabilization: 0.2% Triton X-100 in PBS
  • Blocking: 3-5% BSA in PBS
  • Primary antibody: Cell-validated
  • Secondary antibody: Fluorophore-conjugated
  • Nuclear stain: DAPI (1 μg/ml)
  • Mounting medium: Antifade mounting medium

Step-by-Step Procedure

1

Cell Seeding and Growth

  1. Place coverslips in wells of 24-well plate or chamber slides
  2. Sterilize coverslips: Autoclave or soak in 70% ethanol, then UV sterilize
  3. Optional coating:
    • Add poly-L-lysine (0.1 mg/ml) for 30 minutes
    • Rinse with sterile PBS
  4. Seed cells at appropriate density to reach 50-70% confluence at time of fixation
  5. Grow cells under normal culture conditions
  6. Apply treatments as required by your experiment

Critical Timing

Fix cells when they reach 50-70% confluence. Over-confluent cells may detach during staining.

2

Fixation

For PFA Fixation (Most Common):

  1. Remove culture medium carefully (do not disturb cells)
  2. Gently rinse once with warm PBS (37°C)
  3. Add 4% PFA in PBS (pre-warmed to room temperature)
  4. Incubate for 15 minutes at room temperature
  5. Work in fume hood

For Methanol Fixation:

  1. Remove culture medium
  2. Rinse once with PBS
  3. Add ice-cold 100% methanol
  4. Incubate for 5-10 minutes at -20°C
  5. No permeabilization needed

Verification

After fixation, cells should remain attached to coverslips. Check under microscope if needed.

3

Washing

  1. Remove fixative
  2. Wash 3 times with PBS, 5 minutes each
  3. Keep coverslips in wells during washing
  4. Use gentle pipetting to avoid dislodging cells
4

Permeabilization

Skip if using methanol fixation.
  1. Add 0.2% Triton X-100 in PBS
  2. Incubate for 10 minutes at room temperature
  3. For nuclear antigens: use 0.5% Triton X-100 for 15 minutes
  4. For membrane proteins: use 0.1% Triton X-100 for 5 minutes
5

Blocking

  1. Add 3-5% BSA in PBS
  2. Incubate for 30-60 minutes at room temperature
  3. Do not wash - proceed to primary antibody
6

Primary Antibody

  1. Dilute primary antibody in blocking solution
  2. Add 100-200 μl to each coverslip
  3. Incubate overnight at 4°C (recommended) or 1-2 hours at room temperature
  4. Keep in humidified chamber to prevent drying
  5. Protect from light
7

Washing

  1. Wash 3 times with PBS, 5 minutes each
  2. For high background: use PBS with 0.1% Tween-20
  3. Keep coverslips in wells during washing
8

Secondary Antibody

  1. Dilute secondary antibody in blocking solution
  2. Add 100-200 μl to each coverslip
  3. Incubate for 1 hour at room temperature in the dark
  4. Protect from light throughout
9

Final Washing

  1. Wash 3 times with PBS, 5 minutes each
  2. Final wash should be thorough
10

Nuclear Counterstaining

  1. Add DAPI (1 μg/ml) in PBS
  2. Incubate for 5 minutes at room temperature
  3. Wash once with PBS (5 minutes)
11

Mounting

  1. Place coverslip cell-side down on drop of mounting medium on slide
  2. Gently press to remove air bubbles
  3. Seal edges with nail polish (optional)
  4. Store at 4°C in the dark until imaging
12

Imaging

  1. Allow mounting medium to cure for 30 minutes
  2. Image using fluorescence microscope
  3. Use appropriate filter sets for your fluorophores
  4. Capture images immediately or store at 4°C

Expected Results

  • Cells remain attached and well-spread
  • Specific fluorescence signal at target location
  • Clear nuclear staining (if DAPI used)
  • Low, uniform background

Cell-Specific Considerations

Suspension Cells

For suspension cells:

  1. Fix cells in suspension
  2. Centrifuge and resuspend in each solution
  3. After secondary antibody, cytospin onto slides
  4. Mount directly on slides

Sensitive Cells

For cells that easily detach:

  • Use chamber slides instead of coverslips
  • Reduce washing times
  • Use gentler pipetting
  • Consider using methanol fixation

Troubleshooting

If cells detach during staining:

  • Reduce washing intensity
  • Use chamber slides
  • Check coverslip coating
  • See Troubleshooting Guide for more solutions