1. MGH-Boston, Department of HistoPathology
Immunohistochemistry:  The Art of Disease Diagnosis, drug development and biological research
MGH-Boston, Department of HistoPathology

2. ImmunoHistoChemistry
Introduction and History of IHC.
How IHC works in a clinical lab (the big picture).
Clinical application for medical diagnosis.

3. What is Immunohistochemistry (IHC)?
Immunohistochemistry (IHC): The process of detecting antigens (e.g. proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.
IHC takes its name from the roots "immuno", in reference to antibodies used in the procedure, and "histo," meaning tissue (compare to immunocytochemistry). The procedure was conceptualized and first implemented by Albert Coons in 1941.
Used in the diagnosis of abnormal cells (cancerous tumors or cell death (apoptosis).
Used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue.
Visualizing an antibody-antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyze a color-producing reaction (immunoperoxidase staining).

4. Introduction to Immunohistochemistry (IHC):
Immunohistochemistry (IHC) combines histological, immunological and biochemical techniques for the identification of specific tissue components by means of a specific antigen/antibody reaction tagged with a visible label. IHC makes it possible to visualize the distribution and localization of specific cellular components within a cell or tissue.

5. Introduction to Immunohistochemistry (IHC):
Common Methods of protein detection:
ELISA
Gel Electrophoresis
Western blot
Immunoprecipitation
Spectrophotometry
Enzyme assays
X-ray crystallography
NMR
Immunohistochemistry

6. Introduction to Immunohistochemistry (IHC):
Immunohistochemistry – what’s good about it?
Antibodies bind to antigen in specific manner
Gives you a spatial location
Can be used to locate particular cells and proteins
Can be used to identify cellular events – e.g.apoptosis

7. History of Immunohistochemistry (IHC):
The principle has existed since the 1930s.
Started in 1941 when Coons identified pneumococci using a direct fluorescent method.
Indirect method
Addition of horseradish peroxidase
Peroxidase anti-peroxidase technique in 1979
Use of Avidin & Biotin complex in early 1980’s

8. What cellular antigens can we target?
Cytoplasmic
Nuclear
Cell membrane
Lipids
Proteins

9. Important considerations for IHC
Antibody selection
Fixation used
Sectioning (4-5µm)
Antigen Retrieval
≈pH 6.0 (Low)
≈pH 9.0 (High)
Blocking (clean background)
Controls (Strong positive and Negative)
Multiple labeling.
You actually need to care about all this now because it may affect how you harvest your samples !

10. Options for antibodies that will affect your results
Monoclonal vs. Polyclonal.
Mouse or Rabbit
Raised against whole molecule, N-terminus, C- terminus, specific amino acids
Ascites, supernatant, serum

11. General Antibody Structure and Classification:
The two variable regions recognise bind to antigen (parts of invading bacteria) leading to the invading bacteria being destroyed. Antibodies produced in the body are polyclonal, because each one has a different variable region and can target a different antigen (until a threat is realised in which case they massively overproduce the relavent antibody). The two variable regions recognise bind to antigen (parts of invading bacteria) leading to the invading bacteria being destroyed. Antibodies produced in the body are polyclonal, because each one has a different variable region and can target a different antigen (until a threat is realised in which case they massively overproduce the relavent antibody).

12. Monoclonal v. polyclonal
Mouse or rabbit hybridoma
Tends to be ‘cleaner’
Very consistent batch-to-batch
More likely to get false negative results
Polyclonal
Many different species
Non-specific reactivity
Different avidity & affinity batch-to-batch
More likely to have success in an unknown application

13. Fixation Aldehyde Frozen 10% NBF (Labs standard)
4% formaldehyde with PBS buffer
2% formaldehyde with picric acid and PBS
The paraformaldehyde paradox
Immersion v. transcardial perfusion
24-72 hours
Many others
Best for good architecture
Frozen
LN2
With or without sucrose
OCT
Fix with acetone or methanol (fix by coagulation, also permeabilizes)
Best for cell membrane antigens, cytokines
Formaldehyde fixes by formation of hydroxymethyl adducts on reactive side chains of proteins. Once sufficient adducts are formed, they slowly cross-link to stabilize the proteins in a gel-like formation. At room temperature, it takes approximately 24 hours for maximal binding of formaldehyde to occur and hence all the adducts to form. These initial adducts, and any initial cross-links formed, are unstable and easily reversed. For tissues fixed for 24 hours, the cross-links are largely reversed by washing ( in water or 70% EtOH) after a few hours. It probably takes at least 5-7 days for most of the cross-links to form, and a small amount of cross-linking still continues over time. Even after fixation for 7 days or more, the cross-links can still be reversed by prolonged washing. That's great for IHC, since cross-links can be reversed by HIER (which is just washing at elevated temperatures!).

14. Fixation Estrogen receptor (ER) A. 3 hours in 10% NBF
B. 8 hours in 10% NBF
Influence of fixation time, fixation temperature, and trypsin treatment on uPA immunoperoxidase staining of paraffin-embedded specimens of human ductal breast cancer tissue. Specimens were formalin fixed at 4° C for 1 hour (Panels a, f, g), 8 hours (Panel b), and 24 hours (Panel c) or at room temperature for 8 hours (Panel d) and 24 hours (Panel e), and immunostained with polyclonal antibodies to uPA (pAb2, 5 g/ml). Most sections were briefly predigested with trypsin (Panels a–f), although this treatment was omitted for one of the sections (Panel g). Note that when fixation was performed at 4° C, the immunostaining produced is strong in specimens fixed for 1 hour and 8 hours (Panels a, b, f), but barely detectable after fixation for 24 hours (Panel c). At room temperature fixation for 8 hours results in weak staining (Panel d), and fixation for 24 hours results in no immunostaining (Panel e). When trypsin predigestion is omitted, there is little or no uPA immunostaining, even of sections formalin fixed for 1 hour at 4° C (Panel g). Note that the immunostaining in all sections appears in the stroma and not in the cancer cells and that the strongest immunostaining is present in the fibroblast-like cells surrounding the tumor cell islands (arrows in Panel a). Bars in Panels a–e: 30 m; Panels f–g, 50 m
LCA (CD45) on Tonsil staining of lymphocytes.
3 hours in 10% NBF.
8 hours in 10% NBF.

15. Antigen retrieval HIER PIER
Use MW/steamer/pressure cooker ~ 20 minutes, slow cool
Citrate 6.0
Tris-EDTA 9.0
EDTA 8.0
Must determine for each new antibody/antigen target
PIER
Proteinase K
Trypsin
Pepsin
Pronase,etc.
Destroys some epitopes
Bad for morphology

16. Blocking Background staining Specific Non-specific
Polyclonal antibodies – impure antigen used
Inadequate fixation – diffusion of antigen – often worse in center of large block
Non-specific
Non-immunologic binding – usually uniform
Endogenous peroxidases
Endogenous biotin

17. IHC direct and direct staining:
In Situ Hybridization (ISH) work with DNA and RNA staining by denaturalization of both band of DNA (DNA Probes) or to a single band of RNA (RNA probes) in order to find a specific mutation or target gene in the cells. RNA probes being more sensitive.

18. IHC: Working up and troubleshooting, Non-specific staining.
Before block
After block

19. Adequate Controls for IHC testing:
Positive control
Best is tissue with known specificity
Negative control
Best is IgG from same species immunized against non-biologic molecule – e.g., BRDU when no BRDU is present in tissue
Can also use non-immunized serum from same species

20. How IHC works in a clinical lab (the big picture).
Microtomy, Control, Oven 60º C.
Loading Kits, antibody, Slides.
Dehydrate and Quality Control.

21. Clinical application for medical diagnosis.
Breast Carcinomas (Her2 Stains):
Colon Carcinomas (MMR stains):

22. ImmunoHistoChemistry (IHC) Laboratory Team