All-in-One Kit for Immunohistochemical Staining for Tissues

The BioModule™ Immunohistochemical (IHC) Staining for Tissue provides qualified reagents and validated protocols to perform highly sensitive and specific immunohistochemical staining of tissues. The key component of Bio-Module™ IHC Staining Unit for Tissues is the SuperPicTure™ Polymer Detection Kit employing HRP detection system to detect mouse, rabbit, rat, or guinea pig primary antibodies bound to tissue antigens. The BioModule™ IHC Staining for Tissues is designed for use with frozen and formalin-fixed, paraffin-embedded tissues.

In addition to the immunohistochemical staining reagents, the BioModule™ IHC Staining Unit for Tissues also includes several key reagents for peroxidase quenching, counterstaining, and mounting.
 
BioModule™ Units for Gene Expression Profiling

The Bio-Module™ IHC Staining Unit for Tissues is one of the several BioModule™ Units available from Invitrogen for gene expression profiling. Each of the BioModule™ Units for gene expression profiling includes high-quality reagents and validated protocols with relevant controls for each step of the workflow (see below). Each unit is designed to provide an integrated workflow that allows you to perform various steps seamlessly during expression analysis. Gene expression profiling comprises multiple steps employing various technologies such as microarray analysis or quantitative PCR (qPCR) for analysis at the nucleic acid level; western immunodetection and immunohistochemistry for analysis at the protein level; and RNAi for functional analysis. 

IHC

Immunohistochemical (IHC) Staining allows you to detect antigens in a tissue fixed on a glass slide. The BioModule™ IHC Staining Unit utilizes indirect staining method to detect antigens and includes the following steps:
 

1. Specific antigen on the tissue binds to unlabeled primary antibody.


2. Labeled secondary antibody conjugated to an enzyme (HRP) binds to the antigen-antibody complex, thus amplifying the signal greater than direct staining methods.


3. Enzyme (HRP) forms a colored (brown), insoluble precipitate at the antigenic sites in the presence of a substrate (H₂O₂) using a chromogen, DAB (3, 3'-diaminobenzidine) that is easily visualized with light microscopy.  IHC staining is simple and easy to perform at the benchtop without the need for any specific instrumentation and allows you to detect antigens in context of tissue morphology. The colored precipitate from DAB is stable for several years providing a permanent record.

 
SuperPicTure™ Polymer Detection Kit

The SuperPicTure™ Polymer Detection Kit features Zymed’s proprietary and enhanced HRP polymer technology to provide excellent sensitivity, high specificity, and a faster protocol than conventional immunohistochemical staining methods. The enhanced HRP polymer technology provides intense nuclear, cytoplasmic, and membrane antigen staining compared to other polymer systems which produce weak nuclear staining due to compromised cell penetration. The SuperPicTure™ Polymer Detection Kit includes all reagents for immunohistochemical staining including buffer, HRP Polymer Conjugate, DAB chromogen, and substrate.
 
System Components

The BioModule™ IHC Staining Unit includes:

• SuperPicTure™ Polymer Detection Kit for highly sensitive and specific immunohistochemical staining of tissue samples using HRP.
• Peroxo-Block™, a specific inhibitor of endogenous peroxidase activity, to effectively eliminate endogenous peroxidase activity without interfering with specific immunostaining.
• Antibody Diluent for diluting primary antibodies. 
• PBS Powder and 50% Tween-20 to prepare washing buffer.
• Mini PAP pen draws a water repellent circle around slide mounted tissue, preventing any wasting of valuable reagents by keeping the liquid pooled in a single droplet.
• Hematoxylin Counterstain Reagent for brilliant staining of nuclei without interfering with the chromogen signal.
• Histomount™, an organic based mounting medium, for immunohistochemical procedures. 

Introduction

General guidelines for using the BioModule™ IHC Staining Unit for Tissues are described in this section. Review the information in this section prior to performing immunohistochemical staining to obtain the best results.

Most of the reagents included with the BioModule™ IHC Staining Unit for Tissues are supplied as ready-to-use solutions without the need for any preparation or dilution and are supplied in a dropper bottle that allows easy dispensing of reagents. We do not recommend diluting the reagents beyond the concentration supplied or removing the reagents from dropper bottles. Do not pipette reagents directly from the bottle.

If you are performing immunohistochemical staining using mouse or rat primary antibodies on mouse or rat tissues, respectively, we recommend that you use HistoMouse™-Max Kit to prevent background problems and obtain best results.

Starting Material

The BioModule™ IHC Staining Unit for Tissues is designed for use with frozen or formalin-fixed paraffin embedded tissue sections mounted on slides using primary antibodies.

Based on your starting sample material, you may need to perform some sample preparation steps, prior to staining the tissue sections.

Fixatives

Appropriate tissue and antigen fixation is required to obtain reproducible performance and reliable interpretations.

Suitable fixatives for most antigens of clinical significance include 10% neutral buffered formalin, B5, Bouin’s, Zinc formalin or alcohol-base fixatives. Formalin-fixed tissues post-fixed in B5 before paraffin embedding may show improved stain.

Cell smears prepared from body fluids should be made to assure a monolayer of cells. Multilayers of cells can trap staining reagents and interfere with the interpretation of results. Fix smears immediately after preparation. Depending on the properties of the antigen, cell smears are usually stable for 1-2 weeks when stored at 4°C.

Primary Antibody

The BioModule™ IHC Staining Unit for Tissues is compatible for use with any mouse, rabbit, guinea pig, and rat primary antibodies. A large variety of high-quality antibodies including the Zymed® Antibodies is available from Invitrogen for use in immunohistochemistry. Prediluted 2nd Gen primary antibodies that are ready-to-use and titered for use with the SuperPicTure™ Polymer Detection Kit are also available. 2nd Gen primary antibodies contain a general protein blocker eliminating the need for a separate blocking step.

Polyclonal or monoclonal antibodies can be used with the kit. For higher specificity, we recommend using monoclonal antibodies. Select antibodies that can detect low antigen levels and can recognize epitopes from formalin-fixed paraffin embedded tissues.

The optimal antibody concentration for use with immunohistochemistry is usually recommended by the antibody manufacturer or you may determine the optimal concentration using a checkerboard titration experiment.

Peroxidase Quenching

Since the BioModule™ IHC Staining Unit for Tissues utilizes HRP detection system, tissues exhibiting high endogenous peroxidase activity will cause high background. Usually the endogenous peroxidase activity is inhibited (quenched) using H₂O₂ pre-treatment. The BioModule™ IHC Staining Unit for Tissues includes Peroxo-Block™, a ready-to-use efficient peroxidase inhibitor.

Because different tissues have different levels of endogenous peroxidase activity, you may need to perform the peroxidase quenching step, only if your tissue exhibits high endogenous peroxidase activity.

To assess the endogenous peroxidase activity in your tissue, hydrate the slide containing tissue sections in PBS. Apply the DAB substrate, incubate for 10 minutes, and wash with distilled water. Examine the slide under the microscope to see any staining. If there is staining, you need to perform the peroxidase quenching step using Peroxo-Block™.

Epitope Retrieval

Antigens that are masked by formalin fixation and embedding procedures can be retrieved using standard proteolytic or heat treatment procedures prior to performing the immunohistochemical staining. Some antibodies require epitope retrieval as recommended by the antibody manufacturer while staining with some antibodies is enhanced by epitope retrieval.

Appropriate Controls

When performing immunohistochemical staining, it is important to include proper positive and negative controls to help evaluate your results.

We recommend including the following three control slides that are necessary for interpreting results. Additional controls can be added based on the experimental design.

Positive Tissue Control

• A specimen processed in the same way as the unknown and contains the antigen to be stained.  

Reagent Control

• An additional slide that is treated with a non-immune serum or isotype control antibody that matches the isotype of the experimental antibody instead of the same concentration of primary antibody. Any staining observed on the specimen is probably due to non-specific protein binding or non-specific binding of other reagents.
• Rabbit and mouse primary antibody isotype controls are available from Invitrogen.

Negative Control

• A specimen processed in the same way as the unknown but does not contain the antigen to be stained (optional).  

Caution:  Handle human origin products according to biosafety practices as outlined for your institution. 
 

An experimental workflow for using the BioModule™ IHC Staining Unit with formalin-fixed paraffin embedded tissues is shown below.




Materials Needed
 
Materials supplied with the BioModule™ IHC Staining Unit for Tissues and User Supplied materials are listed below. Ordering information is above. 

• Fresh, frozen tissue, or paraffin embedded tissue of choice
• Microtome and cryostat for tissue sectioning
• Pre-coated slides (see below)
• Coplin jars or equivalent
• PBS (supplied with the kit)

• OCT® Compound
• Chilled 100% acetone for fixing

• Xylene
• Graded series of ethanol (80%, 95% and 100% ethanol)
• 55ºC oven

1. Chill 100% acetone at 4ºC (required for Step 7, below).


2. Obtain fresh tissue.


3. Snap freeze fresh tissues in cryomolds containing OCT® (Optimal Cutting Temperature) compound (a solution of glycols and resins which provides an inert matrix for sectioning). Store frozen tissue blocks at -70ºC until you are ready for tissue sectioning.


4. For sectioning, allow the frozen tissue block to equilibrate to the cryostat temperature.


5. Cut 4-6 µm cryostat sections and mount on coated glass slides.


6. Dry tissue sections at room temperature for 30 minutes. If desired, store slides at -70ºC before fixing. If slides are stored at -70ºC, warm the slides to room temperature before the fixing step.


7. Place the slides in 100% acetone at 4ºC for 10 minutes to fix the sections.


8. Remove slides from acetone and air dry for 10-30 minutes.


9. Circle each tissue section using the Mini PAP Pen.


10. Store at -70ºC until use or wash the slide in PBS for 10 minutes and proceed immediately to Peroxidase Quenching. 

1. Obtain or prepare the formalin-fixed paraffin embedded sections of choice.


2. Dry slides containing 4 µm formalin-fixed paraffin embedded sections in a 55ºC oven for 2 hours or overnight (do not allow the temperature to exceed 60ºC). Store the slides containing the formalin-fixed paraffin embedded tissue sections at room temperature until needed.


3. Place slides in xylene for 5 minutes at room temperature.


4. Remove slides and place in xylene a second time for an additional 5 minutes.


5. Remove slides and place in 100% ethanol 2 times for 5 minutes each time.


6. Remove slides and place in 95% ethanol for 5 minutes.


7. Remove slides and place in 80% ethanol for 5 minutes.


8. Remove slides and place in PBS for 10 minutes.


9. Drain any excess reagent by tapping the edge of the slide on paper towels and wipe the area near the tissue sections with a laboratory wipe.


10. Circle each tissue section using the Mini PAP Pen.


11. Proceed immediately to Peroxidase Quenching. 

Introduction

Immunohistochemical staining procedure using the SuperPicture™ Polymer Detection Kit is described below.
The total staining time including counterstaining is ~80 minutes.
 
Experimental Outline

For immunohistochemical staining of your samples, you will:
 
  1.     Perform peroxidase quenching step using the Peroxo-Block™, if your sample exhibits high endogenous peroxidase activity.
 
  2.     Incubate the samples with an appropriate dilution of the primary antibody.
 
  3.     Wash any unbound antibody.
 
  4.     Incubate the samples with HRP Polymer Conjugate.
 
  5.     Wash any unbound polymer conjugate.
 
  6.     Add diluted DAB chromogen to develop the signal.
 
  7.     Perform counterstaining with hematoxylin.
 
  8.     Mount the slides using Histomount™.
 
  9.     Visualize the staining using a light microscope.
 
To obtain the best results, follow these recommendations:

• Perform all steps at room temperature, unless a different temperature is specified.
• Always wear protective clothing, safety glasses, and gloves while handling the slides and reagents.
• Do not allow the slides to dry once the staining procedure has started.
• To avoid dislodging the tissue cores, handle the slides gently, especially during the washing steps.
• Perform the staining procedure using Coplin jars, staining dishes or automated instruments.
• Perform all incubation steps with various solutions in a humidified chamber.
• Be sure to run appropriate controls.
• Determine the optimal titer of the antibody through serial dilutions.
• Apply enough reagents to cover the tissue during reagent incubation steps; usually ~100-200 µl.
• Drain any excess reagents after washing by tapping the edge of the slide gently on the jar or on paper towels for a few seconds.

Materials Needed

You will need the following materials:

• Tissue sample
• Primary antibody
• Distilled water
• Xylene or xylene substitutes
• Ethanol (95% and 100%)
• Coplin jars or equivalent
• Humidified chamber
• Coverslips
• Optional: 3% hydrogen peroxide in methanol

For Epitope Retrieval

• Hot plate
• 20X Citrate buffer, pH 6.0
• 20 X EDTA Solution, pH 8.0 (for antibodies that require EDTA instead of citrate buffer)
• Optional: Digest-All™ Kit  for enzymatic digestion

 
The following components are supplied with the kit:

• Peroxo-Block™
• SuperPicTure™ Polymer Detection Kit
• PBS and 50% Tween-20
• Antibody Diluent
• Hematoxylin Counterstain Reagent
• Histomount™
• Mini PAP Pen 

Preparing PBS

PBSPBS (10 mM phosphate buffer, 150 mM NaCl, pH 7.2-7.3) is supplied as a dry powder in the kit. To prepare 1X PBS, dissolve one envelope (package) in 1000 ml distilled water and mix well. Store at room temperature until use.

PBS with 0.05% Tween-20

To prepare 1 L PBS with 0.05% Tween-20, add 1 ml 50% Tween-20 supplied with the kit to 1000 ml 1X PBS. Mix well and store at room temperature until use.

Peroxidase Quenching

Peroxidase quenching is optional. Perform this step if elimination of endogenous peroxidase activity is necessary.

Frozen Tissues

1. Treat frozen tissue slides (Step 9) with 100-200 µl Peroxo-Block™ or enough to cover the tissue section for 45 seconds. Do not incubate for more than 45 seconds.


2. Wash slides 3 times with 1X PBS for 2 minutes, each time.


3. Proceed immediately to Incubating with Primary Antibody.

Formalin-fixed paraffin embedded Tissues

1. Treat formalin-fixed paraffin embedded tissue slides (Step 10) with100-200 µl Peroxo-Block™ or enough to cover the tissue section for 1-2 minutes. 


2. Wash slides 3 times with 1X PBS for 2 minutes, each time.


3. Proceed immediately to Incubating with Primary Antibody, or proceed to Epitope Retrieval Protocol

Peroxo-Block™ works efficiently on many tissues, especially with formalin-fixed paraffin embedded tissues, but with some frozen tissues, you may need to use 3% H₂O₂ to obtain optimal results (better morphology or lower background). To use 3% H₂O₂ for blocking endogenous peroxidase activity, incubate the slides in 3% H₂O₂ in methanol for 10 minutes. Wash slides 3 times with 1X PBS for 2 minutes, each time and then proceed to primary antibody incubation.

Epitope Retrieval Protocol

A sample epitope retrieval protocol developed for some Zymed® Antibodies is described below. The epitope retrieval protocol is used to reverse the loss of antigenicity that occurs with some epitopes in formalin-fixed paraffin embedded tissues. Some Zymed® antibodies may require epitope retrieval protocol (e.g., estrogen receptor, and Ki-67 antibodies), while the staining of many other antibodies may be enhanced by epitope retrieval protocol (e.g., S-100, cytokeratin, and synaptophysin antibodies).

You may use any other epitope retrieval protocol recommended by the supplier of your primary antibody.
 
Heat Induced Epitope Retrieval Protocol

1. Perform this epitope retrieval protocol after Peroxidase Quenching. Note: You may use citrate buffer or EDTA solution depending on the antibody manufacturer’s recommendation.


• To 25 ml 20X Citrate Buffer, pH 6.0 solution, add 475 ml distilled water to obtain a 1X Citrate Buffer, pH 6.0 solution.
• To 25 ml 20X EDTA solution, pH 8.0, add 475 ml distilled water to obtain a 1X EDTA solution, pH 8.0.



2. Based on the reagent that you are using, dilute the reagent to 1X as follows:


3. Place the slides in a slide rack and place the rack in a 1 L glass beaker containing 500 ml diluted citrate buffer (1X), pH 6.0 or diluted EDTA (1X) solution, pH 8.0.


4. Place the beaker with slides on a hot plate. Heat the solution until it boils and continue boiling the solution for 15 minutes.


5. Remove beaker from the hot plate and allow the contents to cool for 25 minutes at room temperature.


6. Rinse slides with 1X PBS and proceed immediately to Incubation with Primary Antibody

Epitope Retrieval Protocol Using Enzymatic Digestion

Brief protocol using Digest-All™ Kit  is described below. The Digest-All™ is a flexible and standardized system for tissue digestion and contains trypsin, ficin, and pepsin proteolytic enzymes. The degree of digestion is based on a standardized 10 minute incubation at 37°C. The trypsin solution is supplied with diluent so that different trypsin concentrations can be made to generate a range of digestion activity between that of Ficin and Pepsin. 

1. Perform this epitope retrieval protocol after Peroxidase Quenching


2. Add the digestion enzyme of choice to your tissue sections.  Ficin and pepsin may be applied directly from the bottle. Trypsin requires dilution: 1 drop Trypsin concentrate to 3 drops Trypsin Diluent. Mix well and apply to tissue sections.


3. Incubate for 10 minutes at 37°C.


4. Wash slides in several changes of 1X PBS and proceed immediately to Incubation with Primary Antibody.

Incubation with Primary Antibody

For best results, primary antibody dilution and incubation times need to be determined empirically and are dependent on sample preparation, antibody affinity, amount of antigen present, and antigen accessibility.  The HRP Polymer Conjugate will react with mouse, rabbit, guinea pig, and rat primary antibodies.
 

1. Dilute primary antibody in Antibody Diluent as recommended by the antibody manufacturer or as determined by a titration experiment. Alternatively, you may use prediluted 2nd Gen primary antibodies.


2. Apply 100 µl or enough of the diluted primary antibody to completely cover the tissue on each section.


3. Incubate the slides in a humidified chamber for 30 minutes.


4. Wash slides 3 times with 1X PBS containing 0.05% Tween-20 for 2 minutes, each time.


5. Proceed immediately to Incubation with HRP Polymer Conjugate, below

 
Incubation with HRP Polymer Conjugate

1. Apply 2 drops (100 µl) or enough of the ready-to-use HRP Polymer Conjugate (Reagent A) to completely cover the tissue on each section.


2. Incubate the slides in a humidified chamber for 10 minutes.


3. Wash slides 3 times with 1X PBS containing 0.05% Tween-20 for 2 minutes, each time.


4. Proceed immediately to Signal Development, below.

 
Signal Development

1. Dilute the 20X DAB Chromogen supplied with the kit in a sterile microcentrifuge tube just prior to use as follows:
   
   TOP
   
   Distilled water                                              1 ml
   Reagent B1                                                  1 drop
   Reagent B2                                                  1 drop
   Reagent B3                                                  1 drop
   
   Mix well. Protect from light and use within 1 hour.


2. Apply 2 drops (100 µl) or enough of the diluted DAB chromogen (prepared in Step 1) to completely cover the tissue on each section.
    
   Caution:   DAB is a known carcinogen; handle with care.


3. Incubate the slides in a humidified chamber for 5 minutes or until the desired signal intensity is reached.
   Incubation time can vary depending on the antibody.


4. Rinse thoroughly with distilled water.


5. Proceed immediately to Counterstaining, below.

Counterstaining

1. Apply 2 drops (100 µl) or enough of the Hematoxylin Counterstain Reagent to completely cover the tissue on each section.


2. Incubate the slides in a humidified chamber for 1-3 minutes.


3. Rinse thoroughly with distilled water.


4. Dip the slide in ammonia water 1X PBS or (0.25% NH3) to blue the nuclei.


5. Rinse thoroughly with distilled water.


6. Dehydrate through increasing concentrations of ethanol (95%,100%) for 10-20 dips each. Clear in 2 changes of xylene or xylene substitutes for 10-20 dips each.


7. Proceed immediately to Mounting, below.

 
Mounting

  1.     Apply 2-4 drops (100-200 µl) of Histomount™ mounting medium to the slide.
 
  2.     Apply a cover slip on the slide.
 
  3.     Allow the medium to dry at room temperature overnight.
 
Microscopy

Evaluate the results by examining the slides using a light microscope at 20x magnification. Interpret the results as described below.

Example of Results
 
An example of results obtained after performing an immunohistochemical staining experiment with the BioModule™ Immunohistochemistry (IHC) Staining for Tissue is shown below.
 
In this experiment, formalin-fixed paraffin-embedded human testis tissue was subjected to the immunohistochemical staining protocol using 2ndGen predilute (ready-to-use) rabbit anti-phospho-MAP kinase ERK1/2 antibody from Invitrogen (cat. no. 08-1389) as described in this unit manual. 
The image shows phospho-MAP kinase-positive cells that are stained brown from the DAB chromogen, displaying the expected nuclear staining pattern.

Introduction

Review the information in this section to troubleshoot your immunohistochemical staining experiments.