1. The basics of immunohistochemistry
Department of Clinical Sciences Faculty Meeting –
The basics of immunohistochemistry

2. Common Methods of protein detection
ELISA
Gel Electrophoresis
Western blot
Immunoprecipitation
Spectrophotometry
Enzyme assays
X-ray crystallography
NMR
Immunohistochemistry

3. 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

4. Introduction
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. History 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

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

7. Identify replicating cells

8. Locate cells that are signaling

9. Locate apoptotic cells

10. Identify activation states

11. Identify different types of cells in a tissue

12. Examine cytoskeletal structure

13. Important considerations for IHC
Antibody selection
Fixation
Sectioning
Antigen Retrieval
Blocking
Controls
Direct method
Indirect method
Immunoenzyme
Fluorescence
Multiple labeling
You actually need to care about all this now because it may affect how you harvest your samples !

14. Options for antibodies that will affect your results
Monoclonal v. Polyclonal
Raised against whole molecule, N-terminus, C-terminus, specific amino acids
Ascites, supernatant, serum

15. General antibody structure
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).

16. 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
Tends to have more non-specific reactivity
Can have very different avidity/affinity batch-to-batch
More likely to have success in an unknown application

17. Make sure your antibody is validated for your application!!!
IF v. IHC with fluorescence
WB, ELISA, IP, etc.

18. Whole molecule or specific portion of epitope?
Very dependent on individual assay

19. Ascites, supernatant, serum?
Differences in affinity/avidity
Ascites – highest affinity
Supernatant next
Serum lowest
Depends on concentration!

20. Fixation Aldehyde Frozen 10% NBF 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!).

21. Plasma urokinase inhibitor – 48 hours fixation v. 7 days fixation
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
Plasma urokinase inhibitor – 48 hours fixation v. 7 days fixation

22. Sectioning Paraffin Frozen
Must heat and process through xylenes and alcohols – ruins some antigens
Most commonly used
BEST if not stored more than two weeks – lose antigenicity after that time
Frozen
Better survival of many antigens
Poor morphology
Poor resolution at higher mag
Special storage
Cutting difficulty

23. 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

24. Improving antibody penetration
Need this for intracellular (cytoplasmic, nuclear) or membrane components when epitope is inside cell membrane
Detergents most popular
Triton-X
Tween
Also decreases surface tension – better coverage
Can’t use for membrane proteins
Acetone/Methanol
Precipitate proteins outside cell membranes- more accessible
Saponin
Punches holes in cell membrane – holes close up when removed

25. 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

26. Non-specific staining
Before block
After block

27. Controls Positive control Negative 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

28. Direct method- primary antibody only
Goat anti-actin labeled with 594
Fluorochrome conjugated primary antibody

29. Indirect method – primary and secondary antibodies
Donkey anti-goat labeled with 488
Goat anti-actin

30. Enzyme linkage indirect method
Flourochrome (488) conjugated streptavidin
Biotinylated donkey anti-goat
Goat anti-actin

31. Multiple Immunofluorescence

32. Multiple Labelling of a Tissue Section

33. Enzymatic detection methods
Brightfield microscope sufficient for analysis of specimens Suitable for tissue analysis at low magnification Resolution of subcellular structures not as good as with fluorescence methods, but can be combined with electron microscopy Unimited shelf life of labelled specimens Substrate reagents often toxic/carcinogenic

34. PAP Method (peroxidase anti-peroxidase method)

35. ABC method

36. SP Method (streptavidin peroxidase conjugated method)

37. Beta-2 toxin for C. perf - DAB

38. Summary IHC = immunology +histology + chemistry
Has strengths and weaknesses
Think about your planned assay before acquiring tissue
Good block, appropriately fixed and sectioned can give you great data
Bad block, inappropriately fixed and sectioned, can give you misleading data and waste money