#7 IHC Double Staining

#7 IHC Double Staining

The DNA of a nucleus directs the actions of the cell by instructing it to manufacture specific proteins.  This “instruction” is sent from the nucleus into the cytoplasm by “messenger RNA” (mRNA).  The mRNA exits the nucleus to bring the “message’” into the cytoplasm, binding with ribosomes to manufacture proteins.  Figure 1 illustrates this principle.

In the #5: In Situ Hybridization (ISH) segment of the series, ISH methods were discussed and described as a technique whereby a single strand of nucleotides can be used as a probe to recognize and bind to its complementary strand of DNA – or RNA – located “in place” within the nucleus – or cytoplasm – of cells present in a tissue section.

The current climate in histology favors the development of these new molecular methods and techniques.  Why?

More and more treatments for patients are being based upon genetic information from the cell.  Pathologists, clinicians and their patients want to obtain as much information as possible from the tissue sections that we prepare in the laboratory.  IHC and ISH can localize proteins and nucleic acids within cell nuclei and cytoplasm, as well as in areas outside of the cell.  Current methodology allows for double and triple staining of different targets in the same tissue section.  This provides extremely valuable information for the pathologist, as single cells can be assessed for the presence or absence of the desired targets within nuclei, cytoplasm and outside of the cell.

A good example of this concept is the current “PIN 4” (prostatic intraepithelial neoplasia) staining procedure for prostate tissue suspected of containing carcinoma cells.  Three different proteins are visualized.  High molecular weight keratin (HMW) identifies inner luminal cells.  P63 protein identifies the basal layer of stratified epithelial cells. These two antibodies are combined in one cocktail and therefore used to identify the basal layers of glandular epithelium within the prostate tissue section.  The third protein is identified by an antibody against P504s, which is also called “racemase”.  Racemase is an enzyme produced by prostate carcinoma cells.

In the double staining method, the HMW / P63 antibody cocktail is applied to the tissue section first.  Then a detection chemistry for DAB staining is applied to result in dark brown staining at the antigens’ sites within the basal cell layer.  Since both proteins identify the same cell type, it is not necessary to know whether one or both antigens are present – simply that these cells are identified as staining dark brown.

After the stain is completed, a second stain procedure is performed on the same section, using a primary antibody against P504s.  For this antibody, a fast red detection chemistry is used such that final stain localization is red.  Red staining within cells indicates that the cell is making P506 protein / racemase and is identified as a cancer cell.  Figure 2 shows an example of this stain procedure.

This is simply one example of many possibilities using double and triple staining procedures.  An excellent paper written by David C. Spaulding describes a specific method for “Simultaneous Detection of Nucleic Acids and Proteins in Pathological Specimens” (1).  The paper describes how to identify specific nucleic acids in the nuclei of cells while, in the same section, identifying specific proteins related to these genes.

Clearly, the “molecular revolution” in pathology is underway and histologists should not only be aware of it, but actively participating in it.

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  1. Spaulding D. C. Applications of Combined HIS and IHC Methods for Simultaneous Detection of Nucleic Acids and Proteins in Pathological Specimens.  Acta Histochem CytoChem, Vol 28, No 1, 83-84. 1995.
  2. Chapman C. M. The Histology Handbook.  Amazon CreateSpace Independent Publishing Platform; 2017.
  3. Chapman C. M., Dimenstein I. B. Dermatopathology Laboratory Techniques.  Amazon CreateSpace Independent Publishing Platform; 2016.
#6 IHC Nuts and Bolts

#6 IHC Nuts and Bolts

Immunohistochemistry Educational Series

In previous installments of the IHC Educational Series, we discussed the specifics of the basic procedures found in the IHC laboratory: immunofluorescence, immunohistochemistry and in situ hybridization. Even though these are different procedures, they all require the following support procedures.
Whenever a new antibody (or probe) is ordered for use by a pathologist for diagnosis using IHC, an antibody validation must be completed which consists of multiple procedures. Initially, analytical sensitivity of the antibody must be performed to determine the appropriate dilution of the antibody, along with the incubation time, conditions and optimal antigen retrieval conditions, if required. The antibody manufacturer will include a product insert sheet which will state the suggested antigen retrieval conditions, primary antibody dilution and incubation time/ conditions; this is where the IHC technician will start.
It is good practice to begin with a dilution series, with the recommended dilution in the middle, bracketed on either side by stronger and weaker dilutions. For example: if the recommended dilution is (1:100), a staining run should be done on sections of the same positive control tissue using dilutions of (1:25), (1:50), (1:100), (1:200) and (1:400) – all at the same incubation time, temperature and antigen retrieval suggested in the insert. The pathologist can then view the slides and determine which dilution results in optimal staining with minimal/ no background stain. After the best dilution is determined, the stain should be repeated using the optimal dilution and conditions on several different tissue types, both positive and negative. This verifies the procedure and measures analytical sensitivity and specificity, as the pathologist can confirm the staining is specific.
Once this is done, the staining protocol should be tested and verified for precision and reproducibility, which actually measures the staining instrument performance. Precision is measured by loading the stainer with serial sections of the selected positive control tissue on one instrument run. All slides should stain identically, which confirms the precision of the stainer.
Reproducibility is measured by staining sequential positive control sections on consecutive instrument staining runs. Again, all slides should stain identically, which confirms that the stain is reproducible, no matter what slide position in the stainer is used and no matter what day or time the stain is run.

Once the procedures of analytic sensitivity, specificity, precision and reproducibility have been successfully completed and signed off by the Medical Director, the antibody is considered validated and ready for use.
Whenever a new lot of antibody is ordered and received, a new Reagent Lot Verification procedure must be completed and signed off by the Medical Director prior to the antibody being used on patient specimens. A new dispenser of the primary antibody should be made up according to the original validation records and used to stain the positive control tissue slide. Ideally, one slide should be stained with the old lot, and another slide stained with the new lot. The Medical Director must view both slides and confirm that the staining is optimal and specific, prior to signing off on the Reagent Lot Verification Form.

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  1. Chapman CM. The Histology Handbook.  Amazon CreateSpace Independent Publishing Platform; 2017.
  2. Chapman CM, Dimenstein IB. Dermatopathology Laboratory Techniques.  Amazon CreateSpace Independent Publishing Platform; 2016.
  3. https://www.ncbi.nlm.nih.gov/probe/docs/techish/
  4. https://www.sciencedirect.com/topics/neuroscience/in-situ-hybridization
  5. Bishop JA et al. Detection of Transcriptionally Active High-risk HPV in Patients With Head and Neck Squamous Cell Carcinoma as Visualized by a Novel E6/E7 mRNA In Situ Hybridization Method.  American Journal of Surgical Pathology, 36(12):1874-1882; 2012.
#5: In Situ Hybridization

#5: In Situ Hybridization

In the previous segments of the IHC Educational Series, immunofluorescence and immunoperoxidase methods were discussed.  In both of these methods, antibodies are used to bind to and localize protein antigens in tissue sections.  Additionally, the differences between the use of polyclonal and monoclonal antibodies had been described.

Another immunohistochemical method is “in situ hybridization”. The term “in situ” means “in the original place”, while “hybridization” refers to a hybrid composed of one strand of DNA with a complementary strand of nucleotides – which is referred to as a probe.  Thus “in situ hybridization” describes a technique whereby a single strand of nucleotides is used as a probe to recognize and bind to its complementary strand of DNA, located “in place” within the nuclei of cells present in a tissue section.

Double stranded DNA is built from simple building blocks of four amino acids: adenine, thymine, cytosine and guanine.  Adenine always binds with thymine and cytosine always binds with guanine.  DNA can “unwind” in the nucleus to provide a template to construct complementary RNA (“transcription”).  The RNA then exits the nucleus to bring the “message’” into the cytoplasm, binding with ribosomes to manufacture proteins.  Figure 1 illustrates this principle.

When double stranded DNA within a tissue section is heated to approximately 95 C in the presence of an appropriate buffer, the strands separate.  Once separated, a nucleotide probe (attached to a label) can be placed on the slide and incubated.  The probe will bind to its complementary strand of DNA.  Upon cooling, the DNA strand will close back up.

Labels, or “reporter molecules” can be radioactive molecules.  However, in histology, usually biotin, digoxigenin or fluorescent labels are used.  If fluorescent molecules are used, the method is referred to as “FISH”: fluorescent in situ hybridization.  The staining results are viewed in a darkfield fluorescence microscope.  If biotin or digoxigenin is used in conjunction with a chromogen label, the method is referred to as “CISH”: chromogenic in situ hybridization.  These slides can be viewed using a standard routine light microscope.  A schematic diagram of the procedure is shown in Figure 2.    

What are these probes recognizing?  Researchers can construct very specific probes that will recognize and bind to very specific DNA sequences within the nuclei of cells.  In this way, specific gene sequences can be recognized within a histologic section of tissue.  The results can help identify cells which are abnormal and/or may eventually give rise to cancer cells.

One such example is the use of probes in the diagnosis of human papilloma virus (HPV) in head and neck cancers.  The literature offers information that the presence of E6/E7 viral oncogenes in biopsies is evidence of the presence of high-risk HPV.  HPV biomarker detection reagents can be used to demonstrate these oncogenes in formalin fixed paraffin embedded tissue sections.  Additionally, these probes can be directed against the mRNA contained in the section.  Positive staining indicates the presence of transcriptionally active mRNA, providing increased sensitivity of the method.  This activity can be demonstrated even when the actual viral load is very low.  Figure 3 shows positive in situ hybridization staining for HPV low risk strains.

Clearly, one aspect of the future of histology is down the path of molecular biology.  Information can now be obtained regarding gene location in the nucleus, and mRNA found in the cytoplasm.  This information is valuable in helping clinicians to formulate prognoses and treatments for their patients.

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#4: Antibodies

#4: Antibodies

In the previous units of this series, the immunohistochemical (IHC) methods of immunofluorescence and immunoperoxidase were described and discussed.  These methods use antibodies to localize cellular proteins in tissue sections, which can then be visualized in either a dark field fluorescence microscope or routine bright field microscope.  It is important to understand antibody structure and function in order to fully appreciate these immunohistochemical (IHC) techniques.

Antibodies are proteins made by mammals in response to foreign “invaders” such as bacteria, viruses and other protein elements.  Each antibody is made in response to specific antigenic determinants on those proteins, that are called antigens.  Each antibody is made by specialized B cells, which divide and form clones which provide long term “memory” of the antigen.  This makes up part of the mammalian immune system which provides protection against such “invaders”.

Antibodies themselves are made of four polypeptide chains: two identical copies of a heavy chain and two identical copies of a light chain, which contain a variable region.  The variable region binds a unique, specific epitope region on the antigen protein.  The immunoglobulin class of an antibody is defined by the heavy chain makeup, while the light chain is classified as a kappa or lambda type.  Figure 1 shows a schematic diagram of a typical antibody.  Figure 2 shows the “protein” configuration of an antibody.

 Figure 3 shows the protein configuration of an antigenic protein.  The five different colors depict five different antigenic determinants on the protein.  Each determinant would be recognized by a different antibody clone.  If an antibody preparation is “monoclonal”, it contains only one of the antibody clones.  In this scenario, the monoclonal preparation would contain only the red antibody (i.e. red “Y”).  If an antibody preparation is “polyclonal”, it contains many antibody clones – each directed to a specific antigenic determinant on the protein.  In this scenario, the preparation would contain all of the different colored antibodies, as shown in the diagram.

When performing IHC methods, the histotechnician and pathologist must determine the best type of antibody for the project.  There are many vendors which provide many primary antibody preparations for use in IHC.  Some are provided in concentrated “neat” forms, which must be optimized in terms of concentration and antigen retrieval conditions.  Other primary antibodies are provided in “ready to use” form: already diluted and optimized for the specified procedural conditions.  Whether to use a monoclonal or polyclonal antibody is up to the pathologist.

Once the decision has been made on which primary antibody to use, the procedure must be optimized by the laboratory and validated prior to utilization on patient specimens.  This is done by running experimental staining procedures using a positive control tissue.  When the pathologist is able to visualize the proper staining pattern and intensity in the positive control tissue, the stain is ready for use in the laboratory.  It is important to remember that the stain procedure must be validated each time a new lot of primary antibody is ordered and received – even if it is the same.  It is possible that lot-to-lot variation can occur and may have an effect on the final stain.

Figure 1.
Schematic representation of an antibody.

Figure 2.
Antibody, space-filling model. Atoms are coloured according to convention.

Figure 3.

3D configuration of a protein, showing different colored antigenic determinants within the protein.  The different antibody clones are shown as corresponding colored “Y”’s.

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  1. Chapman CM. The Histology Handbook.  Amazon CreateSpace Independent Publishing Platform; 2017.
  2. Chapman CM, Dimenstein IB. Dermatopathology Laboratory Techniques.  Amazon CreateSpace Independent Publishing Platform; 2016.
  3. https://www.ncbi.nlm.nih.gov/books/NBK26884/ B cells and antibodies.
  4. https://www.dreamstime.com/royalty-free-stock-images-antibody-structure-image26640389
#3: Immunoperoxidase

#3: Immunoperoxidase

In the previous segment, immunofluorescence methods were discussed.  These methods use antibodies labelled with fluorescein to directly localize cellular proteins in tissue sections, which can then be visualized in a dark field fluorescence microscope.  Despite the advantages of this method, science is always in search of newer methods to gain more knowledge.  Thus in 1970 Sternberger et al (1970) reported an improvement of Graham’s method (1965) using horse radish peroxidase enzyme (HRP) labelled antibodies to localize antigens and visualize them in the routine light microscope – bringing the method “to light” as one might describe.  Now researchers and pathologists could see protein localization within the histology of formalin fixed paraffin embedded tissues viewed using a routine brightfield microscope.  This provided vast improvements over the immunofluorescence method.

Figure 1 shows a schematic representation of Sternberger’s indirect immunoperoxidase method.  Starting at the bottom, a primary antibody (purple) directed against the antigen in question located in the tissue section (blue triangle) is applied to the microscope slide which has been deparaffinized and hydrated.  Once the primary antibody incubation is complete, the slide is washed with buffer, and then a secondary antibody is added (tan).  The secondary antibody is conjugated to horse radish peroxidase enzyme (red).  After washing, the slide is incubated with a diamino-benzidine (DAB) chromogen solution.  Wherever the peroxidase is located, the DAB precipitates out.  The final result is dark brown staining at the site of the antigen localization.

Since the majority of research was done on human tissue, one disadvantage of the Sternberger technique was that the secondary antibody was “anti-human IgG”, and therefore might bind to endogenous human IgG present in the tissue section, thereby causing background staining.  To eliminate this possibility, researchers used biotin to label the secondary antibody followed by an avidin-HRP complex.  This decreased most non-specific binding.  However, in tissues high in biotin (i.e. liver) the possibility was now that the avidin could bind non-specifically to that endogenous biotin and cause additional background staining.  This was finally solved by the methods currently used in which polymers are used as the linking reagent and HRP (or other enzyme) carrier.  Figure 2 shows this current method as compared to the original Sternberger method.

In summary, direct immunofluorescence (DIF) techniques use a primary antibody that is directly conjugated to the fluorescein label.  This causes a one-to-one, signal-to-antigen result and must be viewed in a dark field microscope.  The indirect immunoperoxidase of Sternberger, as currently modified using polymers, results in many signals to one antigen, thereby causing the staining to be increased.  This technique, combined with the ability to view the results in a routine brightfield microscope, is a great improvement.

It is important to remember that these techniques must incorporate blocking reagents of some sort, in order to eliminate unwanted non-specific staining in the final slide.  Likewise, the use of high purity antibody preparations also decreases the chances of background staining.  There is even the ability to label more than one antigen in a single tissue section.  Figure 3 shows multiple staining in a prostate needle biopsy exhibited in exquisite fashion.  The use of immunohistochemistry in diagnostic pathology has greatly increased the available information from with to make diagnoses and prognoses.  We will see in the next unit that the method can be refined even further.


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#2 Immunofluorescence

#2 Immunofluorescence

Before we discuss immunofluorescence, we need to know what fluorescence is.  There are certain substances, composed of molecules that will emit light when irradiated by a short wavelength, such as X-rays or ultraviolet (UV) light.  The emitted light is of a longer wavelength which has a lower energy.  Two substances that are used in microscopy that fluoresce are rhodamine and fluorescein.  When rhodamine and fluorescein are irradiated by UV light, they will emit red and green light, respectively.

Fluorescein was first synthesized by Adolf von Bayer in 1871.  Later in 1914 Stanislav von Provazek used fluorescent dyes in conjunction with a fluorescence microscope to enhance autofluorescence of cells and tissues.  Albert Coons and his colleagues were the first to report using fluorescein to label antibodies to localize the corresponding antigens in tissue sections viewed under a fluorescence microscope in 1942.  In 1961 Coons mentioned in a seminar that “the hour of the fluorescent antibody” had started.  This is considered to be the beginning of the era of immunofluorescence staining in biology.

Almost eighty years later, the immunofluorescence technique is still used for basic research and patient diagnosis.  This author used direct immunofluorescence in research experiments to localize histaminase activity at the cellular level in the 1970’s.  Similarly, this author has used direct immunofluorescence for many years in dermatopathology applications for diagnosis of immune diseases.

A schematic diagram depicts how direct immunofluorescence (DIF) is performed (Figure 1).  A specific antibody against a specific protein is obtained by immunizing a mammal such as a rabbit or mouse.  Once the animal is producing circulating antibodies, these can be obtained by taking blood specimens and isolating the desired antibodies.  This antibody preparation now undergoes a biochemical process in which molecules of fluorescein are attached to the antibody.  This “fluorescein labelled primary antibody” is applied to a tissue section on a microscope slide and incubated for a time period to allow the antibody to attach and bind to the specific antigen under study.  After rinsing the slide and coverslipping with aqueous mounting medium, the slide is viewed with a darkfield fluorescence microscope.  The slide is irradiated with UV light.  The cellular locations of specific antibody staining can be seen as bright “apple-green” fluorescence as shown in Figure 2.  Pathologists interpret the various patterns and specificities of stains to determine the patient diagnosis.

A schematic representation of the indirect immunofluorescence (IIF) procedure is shown in Figure 3.  The difference between IIF and DIF is that in IIF, a primary antibody is used alone, followed by a secondary antibody which is labeled with fluorescein.  This two-step staining procedure provides great flexibility in the method.  For example, an entire panel of rabbit primary antibodies can be localized using only one secondary antibody directed against the rabbit species of primary antibody.

For diagnosis of autoimmune diseases, the primary antibody can be obtained from the patient’s  blood serum.  When this IgG fraction binds to the appropriate antigen, it can be visualized using a fluorescein labeled anti-human IgG secondary antibody.  The resulting staining intensity and pattern can be interpreted by the pathologist to determine the diagnosis.

In an evolutionary development of the IIF stain, cytochemical staining of peroxidase activity, as reported by Graham et al. (1965), was subsequently developed as an immunohistochemical approach by Sternberger et al. in 1970.  Sternberger was able to use peroxidase enzyme labelled antibodies to localize antigens and visualize them in the light microscope.  This  resolution has moved from the tissue to the intracellular level, and the technique is now used for subcellular localization in even the smallest cells. However, the basic idea remains unchanged, almost seven decades after its first description.  We shall look at this method in detail in the next part of the series.


Figure 1  Schematic diagram of direct immunofluorescence.


Figure 2  Intercellular immunofluorescence staining in epidermal cells of skin.  Original magnification x 600.


Figure 3 Schematic diagram of indirect immunofluorescence.

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1.Chapman CM. The Histology Handbook.  Amazon CreateSpace Independent Publishing Platform; 2017
2. Chapman CM, Dimenstein IB. Dermatopathology Laboratory Techniques.  Amazon CreateSpace Independent Publishing Platform; 2016
3. Lin CW, Chapman CM, DeLellis RA, Kirley SD. Immunofluorescent staining of histaminase (diamine oxidase) in human placenta. J Histochem Cytochem 26:1021, 1978
4. https://www.nature.com/milestones/milelight/full/milelight02.html
5. Coons, A. H., Creech, H. J., Jones, R. N. & Berliner, E. The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody. J. Immunol. 45, 159–170 (1942) | ChemPort |
6. Sternberger, L. A., Hardy, P. H. Jr, Cuculis, J. J. & Meyer, H. G. The unlabeled antibody enzyme method of immunohistochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-anti-horseradish peroxidase) and its use in identification of spirochetes. J. Histochem. Cytochem. 18, 315–333 (1970)
7. Graham, R. C. Jr, Lundholm, U. & Karnovsky, M. J. Cytochemical demonstration of peroxidase activity with 3-amino-9-ethylcarbazole. J. Histochem. Cytochem. 13, 150–152 (1965) | PubMed | ISI | ChemPort