The basics of immunohistochemistry Department of Clinical Sciences Faculty Meeting – 4-14-11 The basics of immunohistochemistry

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

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

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.

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

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

Identify replicating cells

Locate cells that are signaling

Locate apoptotic cells

Identify activation states

Identify different types of cells in a tissue

Examine cytoskeletal structure

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 !

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

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).

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

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

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

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

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!).

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

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

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

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

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

Non-specific staining Before block After block

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

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

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

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

Multiple Immunofluorescence

Multiple Labelling of a Tissue Section

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

PAP Method (peroxidase anti-peroxidase method)

ABC method

SP Method (streptavidin peroxidase conjugated method)

Beta-2 toxin for C. perf - DAB

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