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Immunohistochemistry (IHC) Protocol for FFPE Tissue Sections

This protocol provides a general workflow for manual chromogenic immunohistochemistry (IHC) of formalin-fixed, paraffin-embedded (FFPE) tissue using HRP-polymer detection and DAB chromogen.

Materials

Important: Antigen retrieval conditions, antibody concentration, and incubation conditions are antibody-dependent. When product-specific IHC conditions are provided on a NeoBiotechnologies product page or datasheet, those conditions should be used in place of the general conditions below.

NeoBiotechnologies antibodies are intended for Research Use Only (RUO) and are not approved for diagnostic use.

Materials

  • FFPE tissue sections mounted on positively charged microscope slides
  • Xylene, Slide Brite, or equivalent clearing reagent
  • Ethanol: 100%, 95%, and 70%
  • Distilled or deionized water
  • PBS or TBS wash buffer
  • Antigen retrieval buffer, as appropriate for the antibody
  • Tris-EDTA: 10 mM Tris, 1 mM EDTA, pH 9.0
  • Citrate: 10 mM sodium citrate, pH 6.0
  • 3% hydrogen peroxide
  • Protein blocking solution
  • Primary antibody
  • Species-appropriate HRP-polymer detection reagent
  • DAB chromogen/substrate
  • Hematoxylin
  • Bluing reagent
  • Permanent mounting medium
  • Coverslips

Use appropriate laboratory PPE and handle solvents, DAB, and other reagents according to their current Safety Data Sheets.

1. Slide Preparation and Deparaffinization

  1. Cut FFPE tissue sections at approximately 4–5 µm and mount on positively charged slides.
  2. Bake slides at 60 °C for 15–30 minutes, or according to the laboratory’s validated procedure, to promote tissue adhesion and soften the paraffin.
  3. Deparaffinize the sections:
    • Xylene or Slide Brite: 3 changes × 5 minutes
    • 100% ethanol: 2 changes × 3–5 minutes
    • 95% ethanol: 2 changes × 3–5 minutes
    • 70% ethanol: 1 change × 3–5 minutes
    • Distilled water: 2 changes × 3–5 minutes

Do not allow tissue sections to dry at any point after rehydration.

2. Antigen Retrieval

Antigen retrieval is antibody-dependent. Refer to the product-specific IHC conditions whenever available.

For antibodies validated using high-pH heat-induced epitope retrieval (HIER), a commonly used NeoBiotechnologies manual condition is:

  • 10 mM Tris, 1 mM EDTA, pH 9.0
  • Heat sections to approximately 95 °C for 45 minutes
  • Allow slides to cool at room temperature for approximately 20 minutes

For antibodies requiring low-pH retrieval, use an appropriate citrate-based retrieval buffer, typically 10 mM citrate, pH 6.0.

Some antibodies perform optimally with no antigen retrieval or require alternative retrieval conditions. The antibody-specific product page or datasheet should take precedence over this general protocol.

Following retrieval, wash slides 3 × 3–5 minutes in PBS or TBS.

3. Endogenous Peroxidase Blocking

  1. Completely cover the tissue section with 3% hydrogen peroxide.
  2. Incubate for 10 minutes at room temperature.
  3. Wash slides 3 × 3–5 minutes in PBS or TBS.

4. Protein Blocking

  1. Apply an appropriate serum-free protein block or other validated blocking reagent.
  2. Incubate for approximately 10–30 minutes at room temperature in a humidified chamber.
  3. Drain excess blocking reagent without allowing the tissue to dry.

Follow the blocking reagent manufacturer’s instructions when they differ from the general conditions above.

5. Primary Antibody

  1. Dilute the primary antibody in an appropriate antibody diluent according to the product-specific recommended concentration or dilution.
  2. Completely cover the tissue section with diluted antibody.
  3. Incubate under the conditions specified for the individual antibody.

A commonly used NeoBiotechnologies manual IHC condition for many antibodies is 1–2 µg/mL for 30 minutes at room temperature. This should be treated as a starting condition only when antibody-specific conditions are unavailable.

  1. Wash slides 3 × 3–5 minutes in PBS or TBS.

6. HRP-Polymer Detection

  1. Apply a species-appropriate HRP-polymer detection reagent compatible with the primary antibody.
  2. Incubate for approximately 30 minutes at room temperature, or according to the detection reagent manufacturer’s instructions.
  3. Wash slides 3 × 3–5 minutes in PBS or TBS.

7. DAB Chromogen Development

Prepare DAB immediately before use according to the manufacturer’s current instructions.

  1. Apply sufficient DAB working solution to completely cover the tissue section.
  2. Develop for approximately 5 minutes, or until the desired staining intensity is reached.
  3. Rinse thoroughly in distilled or deionized water.

DAB development time may require optimization depending on antigen abundance, primary antibody concentration, and detection system.

8. Hematoxylin Counterstaining

  1. Counterstain with hematoxylin for approximately 30 seconds to 2 minutes, depending on the formulation and desired nuclear staining intensity.
  2. Rinse thoroughly in running tap water.
  3. Apply bluing reagent according to the manufacturer’s instructions.
  4. Rinse again in tap water followed by distilled water.

The counterstain should provide clear nuclear definition without obscuring the DAB signal.

9. Dehydration, Clearing, and Mounting

Dehydrate the slides through graded ethanol:

  • 70% ethanol
  • 95% ethanol
  • 100% ethanol, two changes

Clear in 2–3 changes of xylene or equivalent clearing reagent.

Apply a compatible permanent mounting medium and coverslip.

Allow the mounting medium to cure before imaging or long-term storage.

Controls

Appropriate controls should be included with each IHC experiment.

Positive control: Use tissue known to express the target antigen to confirm that the staining system and antibody are functioning as expected.

Negative reagent control: Process a comparable tissue section under identical conditions while omitting the primary antibody. This control helps identify staining produced by the detection system or other reagents.

Known negative tissue: When available, tissue known not to express the target can provide additional evidence of staining specificity.

Controls should undergo the same fixation, processing, retrieval, detection, and staining conditions as the experimental samples whenever possible.

Evaluation of IHC Staining

Evaluate staining according to:

  • Expected tissue and cell-type distribution
  • Expected subcellular localization, such as nuclear, cytoplasmic, membranous, or extracellular staining
  • Signal intensity
  • Specific signal relative to background
  • Performance of positive and negative controls

Interpretation should consider both staining intensity and localization. Staining intensity alone should not be interpreted as quantitative protein expression unless the assay has been specifically validated for quantitative or semi-quantitative analysis.

Important Technical Notes

Antigen retrieval and primary antibody concentration are among the most important variables in IHC optimization. Excessive retrieval may damage tissue morphology or reduce staining for retrieval-sensitive epitopes, while insufficient retrieval may result in weak staining.

When optimizing an antibody for a new tissue type or staining platform, evaluate antibody concentration and retrieval conditions systematically rather than altering multiple variables simultaneously.

Automated IHC instruments may use different reagent concentrations, temperatures, retrieval conditions, and incubation times. For automated staining, follow the validated protocol for the specific instrument and detection chemistry.

For Research Use Only. Not for use in diagnostic procedures.

Formalin-fixed, paraffin-embedded human Skin stained with Stratifin Mouse Monoclonal Antibody (CPTC-SFN-2).

Formalin-fixed, paraffin-embedded human Testis stained with 14-3-3E Mouse Monoclonal Antibody (CPTC-YWHAE-1).

Formalin-fixed, paraffin-embedded human Skin stained with 8-oxoguanine Mouse Monoclonal Antibody (CPTC-OGG1-1).

Formalin-fixed, paraffin-embedded human hepatocellular carcinoma stained with TNFAIP3 Mouse Monoclonal Antibody (TNFAIP3/2813).

RE-Hydration Of Tissue Slides

  1. Label the tissue slides according to the protocol. Arrange the slides well in slide racks for easy handling.
  2. Place the slide racks in an oven preheated to 60 °C. Bake the slides for 15 minutes or until the paraffin on the tissue loosens and forms droplets.
  3. Deparaffinize and rehydrate tissue sections by immersing slides through the following series of solutions.
    1. Perform 3 changes of Slide Brite, each for 5 minutes.
    2. Perform 2 changes of 100% ethanol, each for an appropriate duration.
    3. Perform 2 changes of 95% ethanol, each for 5 minutes.
    4. Immerse the slides in 80% ethanol for 5 minutes.
    5. Submerge the slides in 50% ethanol for 5 minutes.
    6. Perform 2 changes of distilled water, each for 5 minutes.
    7. Perform 2 changes of 3% hydrogen peroxide, each for 5 minutes.

Antigen Retrieval

Antigen retrieval can be achieved using BioCare’s Decloacker, which involves boiling the sections at 95 °C for 45 minutes using Tris-EDTA (10 mM Tris with 1 mM EDTA, pH 9.0) OR citrate buffer (10 mM citrate pH 6.0).

Immunohistochemistry

Utilize a hydrophobic marker to establish protective barriers above and below the tissue sections. Exercise caution during this process, as marking too close to the tissue may potentially lead to tissue drying. Therefore, it is advisable to refrain from marking too close to the tissue during this step.

  1. Wash the tissue section twice, 3 times each in PBS.
  2. Incubate the sections with the blocking solution to minimize non-specific binding. Incubation time varies but is typically 30 minutes to 1 hour.
  3. Apply the specific primary antibody to the sections. Dilute the antibody according to the manufacturer’s recommendations. Incubate at the recommended temperature and time (usually overnight at 4 °C or 30 minutes to 2 hours at room temperature).
  4. Wash the sections twice with PBS, 3–5 minutes each, to remove unbound primary antibody.
  5. Apply the appropriate secondary antibody conjugated with an enzyme or fluorophore. Incubate for the recommended time and temperature. Incubate for 30 minutes with secondary antibody, goat anti-mouse / goat anti-rabbit-HRP polymer.
  6. Wash the slides to remove unbound secondary antibody.
  7. Apply the detection system as per your choice. (e.g., DAB for enzyme-linked detection or fluorescent dye for fluorescence detection). For DAB staining, incubate the slides in the DAB solution for 3–5 minutes.
    1. DAB preparation: Mix 950 μL of DAB substrate and 500 μL of DAB chromogen (ScyTek Laboratories) well and use immediately.
  8. Counterstain the nuclei with hematoxylin 3–5 times.
  9. Wash sections with PBS for 3–5 minutes. The final wash should be with distilled water.
  10. Dehydrate through an alcohol series (70%, 95%, and 100% ethanol) and xylene. Mount the slides with a mounting medium and coverslip.
  11. Observe cells under a microscope. 

Analyze the stained tissue sections under a microscope and assess the cellular protein localization and expression levels in tissues, which is vital for research and diagnostics. 

Formalin-fixed, paraffin-embedded human prostate cancer stained with ABCC4 Mouse Monoclonal Antibody (ABCC4/9179). HIER: Tris/EDTA, pH9.0, 45min. 2°C: HRP-polymer, 30min. DAB, 5min.

Formalin-fixed, paraffin-embedded human prostate cancer stained with ABCC4 Mouse Monoclonal Antibody (ABCC4/9019). Inset: PBS instead of primary antibody; secondary only negative control.

IHC analysis of formalin-fixed, paraffin-embedded human kidney. Stained using ACE2/7203 at 2ug/ml in PBS for 30min RT. HIER: Tris/EDTA, pH9.0, 45min. 2°C: HRP-polymer, 30min. DAB, 5min.

Formalin-fixed, paraffin-embedded human bladder stained with Aciculin Mouse Monoclonal Antibody (PGM5/3552) at 2ug/ml. HIER: Tris/EDTA, pH9.0, 45min. 2 °: HRP-polymer, 30min. DAB, 5min.

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