IF Protocol Hub

Indirect Immunofluorescence Protocol - Step-by-Step Procedure

Complete indirect IF protocol using unlabeled primary and fluorophore-conjugated secondary antibodies. Ready-to-use procedure with verification steps.

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

Indirect Immunofluorescence Protocol

Indirect immunofluorescence uses an unlabeled primary antibody followed by a fluorophore-conjugated secondary antibody. This is the most commonly used IF method due to its signal amplification and flexibility.

Advantages of Indirect IF

  • Signal amplification: Multiple secondary antibodies bind to each primary
  • Flexibility: Same primary can be used with different fluorophores
  • Cost-effective: Secondary antibodies are less expensive
  • Higher sensitivity: Generally more sensitive than direct IF
  • Multi-color capability: Easy to combine multiple targets

Pre-Experiment Preparation

Reagents & Materials

Essential Reagents

  • Unlabeled primary antibody: Specific to your target antigen
  • Fluorophore-conjugated secondary antibody: Species-matched to primary
  • Fixative: 4% PFA in PBS (or methanol/acetone)
  • Permeabilization: 0.2% Triton X-100 in PBS
  • Blocking: 3-5% BSA in PBS (or 5-10% normal serum)
  • Nuclear stain: DAPI (1 μg/ml)
  • Mounting medium: Antifade mounting medium

Controls Required

  • Negative control: No primary antibody
  • Isotype control: Isotype-matched control antibody
  • Secondary-only control: No primary, secondary only

Step-by-Step Procedure

1

Sample Preparation and Fixation

Follow standard sample preparation:

Fixation:

  1. Fix with 4% PFA for 15-20 minutes (or methanol/acetone)
  2. Wash 3 times with PBS, 5 minutes each
2

Permeabilization

Skip if using methanol or acetone fixation.
  1. Add 0.2% Triton X-100 in PBS
  2. Incubate for 10-15 minutes at room temperature
  3. Wash once with PBS
3

Blocking

  1. Add 3-5% BSA in PBS (or 5-10% normal serum)
  2. Use serum from the same species as secondary antibody
  3. Incubate for 30-60 minutes at room temperature
  4. Do not wash - proceed to primary antibody

Serum Blocking

Using normal serum from the same species as your secondary antibody helps block Fc receptors and reduces non-specific binding.

4

Primary Antibody Incubation

Antibody Preparation:

  1. Dilute unlabeled primary antibody in blocking solution
  2. Typical dilutions: 1:50 to 1:500
  3. Perform titration to find optimal concentration

Incubation:

  1. Apply 100-200 μl of diluted primary antibody
  2. Incubate overnight at 4°C (recommended) or 1-2 hours at room temperature
  3. Keep in humidified chamber
  4. Protect from light

Verification

After primary antibody incubation, samples should be ready for secondary antibody. Ensure all controls are included.

5

Washing After Primary Antibody

  1. Wash 3 times with PBS, 5 minutes each
  2. For high background: use PBS with 0.1% Tween-20
  3. Ensure complete removal of unbound primary antibody
6

Secondary Antibody Incubation

Antibody Preparation:

  1. Dilute fluorophore-conjugated secondary antibody in blocking solution
  2. Typical dilutions: 1:200 to 1:1000
  3. Ensure secondary is species-matched to primary antibody
  4. Protect from light (fluorophores are light-sensitive)

Incubation:

  1. Apply 100-200 μl of diluted secondary antibody
  2. Incubate for 1 hour at room temperature in the dark
  3. Use aluminum foil or work in dark room
  4. Keep in humidified chamber

Critical

  • Always protect from light during secondary antibody incubation
  • Light exposure will quench fluorescence signal
  • Work quickly and minimize light exposure
7

Final Washing

  1. Wash 3 times with PBS, 5 minutes each
  2. For high background: use PBS with 0.1% Tween-20
  3. Final wash should be thorough
8

Nuclear Counterstaining (Optional)

  1. Add DAPI (1 μg/ml) in PBS
  2. Incubate for 5-10 minutes at room temperature
  3. Wash once with PBS (5 minutes)
9

Mounting

  1. Place drop of antifade mounting medium on slide
  2. Invert sample onto mounting medium
  3. Gently press to remove air bubbles
  4. Seal edges (optional)
  5. Store at 4°C in the dark until imaging
10

Imaging

  1. Allow mounting medium to cure for 30 minutes
  2. Image using fluorescence microscope
  3. Use appropriate filter sets for your fluorophore
  4. Capture images immediately

Expected Results

  • Positive signal: Bright, specific fluorescence
  • Negative control: Minimal fluorescence
  • Isotype control: No specific signal
  • Secondary-only control: No signal (confirms specificity)
  • Background: Low and uniform

Verification Checklist

  • Negative control shows minimal fluorescence
  • Isotype control shows no specific signal
  • Secondary-only control is negative
  • Positive signal is specific and localized
  • Background is low and uniform
  • Signal is bright enough for imaging

Troubleshooting

Weak signal:

  • Increase primary antibody concentration
  • Extend primary antibody incubation time
  • Optimize secondary antibody dilution
  • See IF Troubleshooting Guide

High background:

  • Increase blocking time
  • Use serum blocking
  • Optimize antibody dilutions
  • Increase washing times