Search Thermo Fisher Scientific
Search Thermo Fisher Scientific
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:
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:
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
Reagent Control
Negative Control
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.
Step | Supplied in the kit | User Supplied |
Sample Preparation
|
PBS
|
|
Peroxidase Quenching
|
|
|
Immunohistochemical Staining
|
|
|
Counterstaining
|
|
|
Mounting
|
Histomount
™ |
Coverslips
|
Microscopy
|
Light Microscope
|
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:
Materials Needed
You will need the following materials:
For Epitope Retrieval
The following components are supplied with the kit:
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
Formalin-fixed paraffin embedded Tissues
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
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.
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.
Incubation with HRP Polymer Conjugate
Signal Development
Counterstaining
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.
Problem | Reason | Solution |
Weak or no staining |
Primary antibody was inactive or too dilute
|
Perform staining using a new batch of antibodies.
Be sure to store the antibody at the recommended temperature. Aliquot the antibody into smaller aliquots and store as recommended avoiding repeated freezing and thawing that may result in loss of activity. Check the activity of the antibody using ELISA or Western blotting.
Be sure to perform a titration experiment to determine the optimal antibody concentration.
|
Insufficient incubation times
|
Be sure to perform all incubation steps as indicated in the protocol. You may increase the antibody incubation time to improve staining or increase the substrate incubation time.
| |
Missed steps or steps not performed in the correct order
|
Make sure the staining protocol was followed
| |
Incomplete deparaffinization | Perform the deparaffinization step for a longer time. Use fresh xylene. | |
Epitope not accessible
|
For formalin-fixed paraffin embedded tissues, perform an appropriate epitope retrieval protocol. For frozen tissues, reduce the fixation time.
| |
Substrate prepared incorrectly
| Do not use the 20X DAB chromogen directly from the bottle. Dilute the 20X DAB chromogen.
| |
Tissue sections may have dislodged
|
Always use pre-coated slides to mount tissue sections. We recommend treating the slides with HistoGrip
™ or 0.1% poly-L-lysine, or you may use commercially prepared pre-coated slides.
Handle the slides gently especially during the washing steps.
| |
Samples retain excess liquid after rinsing steps
|
Be sure to remove any excess liquid using paper towels without allowing the tissue sections to dry.
| |
Using sections thicker than normal
|
Thicker sections require longer incubation times for optimal staining.
| |
High background |
Endogenous peroxidase activity not blocked or blocking is incomplete
|
Perform peroxidase quenching step
using Peroxo-Block
™. For some frozen tissues, you may need to use 3% H
2O
2 to obtain optimal results.
|
Incomplete deparaffinization
|
Perform the deparaffinization step for a longer time. Use fresh xylene.
| |
Inadequate washing of slides
|
Perform the recommended washing steps for the appropriate time. Do not skip any washing step.
| |
Longer incubation times used
|
Reduce the incubation time for antibody or substrate.
| |
Over-development of substrate
|
Reduce the incubation time with the DAB chromogen.
| |
High antibody concentration
|
Use diluted primary antibody. Perform a titration experiment to determine optimal primary antibody concentration.
| |
Sections on the slide have dried
|
Do not allow the sections to dry out during the staining protocol. Be sure to add enough reagents to completely cover the sections on the slide (usually 100-200 µl). Perform all incubations in a covered humidified chamber to prevent drying.
| |
Using mouse/rat antibodies on mouse/rat tissues
|
Use HistoMouse
™-Max Kit for best results.
| |
Faint background all over the slide
|
Perform an additional blocking step using 10% normal goat serum blocking solution in PBS for 10 minutes prior to the primary antibody incubation step.
| |
Edge of tissue staining intensely |
Reagents have accumulated under the tissue
|
Increase the washing time after antibody incubations.
|
Bubbles on the slide |
Cover slip not placed properly
|
Avoid trapping any bubbles when placing the cover slip on the slide. If bubbles are already trapped, remove the cover slip by incubating the slide in a 37ºC water bath until the cover slip can be removed easily. Place a clean cover slip on the slide without trapping any bubbles.
|
Negative staining on positive slides |
Specimen was improperly fixed or processed
|
Refer above the recommended sample preparation protocols.
|
Missed primary antibody/HRP Polymer Conjugate incubation steps or steps not performed in the correct order
|
Make sure the staining protocol was followed.
| |
Sections on the slide have dried
|
Do not allow the sections to dry out during the staining protocol. Be sure to add enough reagents to completely cover the sections on the slide (usually 100-200 µl). Perform all incubations in a covered humidified chamber to prevent drying.
| |
Weak or no staining for the antigen in question but there is a precipitate on the slide |
Primary antibody contaminated
|
Use fresh batch of primary antibody. Use sterile pipette tips while handling reagents to prevent contaminating the antibody solution.
|