IF Protocol Hub

IF Protocol for Tissue Sections - Paraffin and Frozen Sections

Complete immunofluorescence protocol for tissue sections including paraffin-embedded and frozen sections. Step-by-step procedure ready for immediate use.

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

IF Protocol for Tissue Sections

This protocol is specifically designed for immunofluorescence staining of tissue sections, including both paraffin-embedded and frozen sections. For general IF procedures, see the General IF Protocol.

When to Use This Protocol

Use this protocol for:

  • Paraffin-embedded tissue sections
  • Frozen tissue sections
  • Formalin-fixed paraffin-embedded (FFPE) tissues
  • Cryosections

Pre-Experiment Preparation

Reagents & Materials

Tissue Section Materials

  • Tissue sections: 4-10 μm thick on charged or poly-L-lysine-coated slides
  • For paraffin sections: Xylene, ethanol series for deparaffinization
  • For frozen sections: Cryostat sections on slides

IF Staining Reagents

  • Antigen retrieval solution: Citrate buffer (pH 6.0) or EDTA (pH 8.0)
  • Fixative: 4% PFA in PBS (if post-fixing)
  • Permeabilization: 0.2% Triton X-100 in PBS
  • Blocking: 3-5% BSA or 5-10% normal serum in PBS
  • Primary antibody: Tissue-validated
  • Secondary antibody: Fluorophore-conjugated
  • Nuclear stain: DAPI (1 μg/ml)
  • Mounting medium: Antifade mounting medium

Step-by-Step Procedure

1

Section Preparation

For Paraffin Sections:

  1. Deparaffinize:
    • Xylene: 2 × 10 minutes
    • 100% ethanol: 2 × 5 minutes
    • 95% ethanol: 2 × 5 minutes
    • 70% ethanol: 2 × 5 minutes
  2. Rehydrate:
    • 50% ethanol: 5 minutes
    • Distilled water: 5 minutes
    • PBS: 5 minutes

For Frozen Sections:

  1. Fix sections (if not pre-fixed):
    • 4% PFA in PBS for 10-15 minutes
    • Or acetone at -20°C for 5 minutes
  2. Wash with PBS: 3 × 5 minutes
2

Antigen Retrieval

Critical for paraffin sections and formalin-fixed tissues.

Heat-Induced Epitope Retrieval (HIER):

  1. Place slides in citrate buffer (pH 6.0) or EDTA (pH 8.0)
  2. Heat in pressure cooker or microwave:
    • Pressure cooker: 15 minutes at full pressure
    • Microwave: 10 minutes at high power (check buffer level)
  3. Cool slides in buffer for 20 minutes
  4. Rinse with PBS

Antigen Retrieval Tips

  • Use citrate buffer (pH 6.0) for most antigens
  • Use EDTA (pH 8.0) for nuclear antigens
  • Some antigens may not need retrieval - test first
3

Permeabilization

  1. Add 0.2% Triton X-100 in PBS
  2. Incubate for 10-15 minutes at room temperature
  3. For nuclear antigens: use 0.5% Triton X-100 for 15 minutes

Frozen Sections

Frozen sections may need less or no permeabilization. Test with 0.1% Triton X-100 first.

4

Blocking

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

Primary Antibody

  1. Dilute primary antibody in blocking solution
  2. Apply 100-200 μl to each section
  3. Incubate overnight at 4°C (recommended) or 2-3 hours at room temperature
  4. Keep in humidified chamber
  5. Protect from light
6

Washing

  1. Wash 3 times with PBS, 5 minutes each
  2. For high background: use PBS with 0.1% Tween-20
  3. Ensure thorough washing
7

Secondary Antibody

  1. Dilute secondary antibody in blocking solution
  2. Apply 100-200 μl to each section
  3. Incubate for 1 hour at room temperature in the dark
  4. Protect from light throughout
8

Final Washing

  1. Wash 3 times with PBS, 5 minutes each
  2. Final wash should be thorough
9

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)
10

Mounting

  1. Add drop of antifade mounting medium
  2. Place coverslip carefully (avoid air bubbles)
  3. Seal edges with nail polish (optional)
  4. Store at 4°C in the dark until imaging

Expected Results

  • Tissue morphology preserved
  • Specific fluorescence signal at target location
  • Clear nuclear staining
  • Low, uniform background
  • No autofluorescence artifacts

Tissue-Specific Considerations

Autofluorescence

Tissue sections may have autofluorescence:

  • Use appropriate controls
  • Consider using TrueBlack or similar reagents to reduce autofluorescence
  • See Troubleshooting Guide for solutions

Thick Sections

For thick sections (10-20 μm):

  • Increase permeabilization time
  • Use higher Triton X-100 concentration (0.5%)
  • Extend antibody incubation times

Troubleshooting

Common issues with tissue sections:

  • High background: Increase blocking time, use serum blocking
  • Weak signal: Optimize antigen retrieval, increase antibody concentration
  • Autofluorescence: Use TrueBlack, optimize filter sets
  • See Troubleshooting Guide for detailed solutions