Immunohistochemistry Microscopic localization of specific antigens in tissues by staining with antibodies labeled with fluorescent or enzymatically processed dyes.

combines histological, immunological and biochemical techniques Antigen: molecular target Antibody Primary: Ig raised against the molecular target Monoclonal Polyclonal Secondary: Ig raised against species of primary antibody; usually conjugated with a reporter Reporter: substance that aids in visualization fluorophore or enzyme Polyclonal Antibodies heterogeneous mixture of antibodies directed against various epitopes of the same antigen more robust, higher cross reactivity Rabbits are frequently the species of choice in polyclonal antibody production due to the ease in maintenance of the animals and relative rarity of human antibodies to rabbit proteins rabbit antibodies precipitate human proteins over a wider range of either antigen or antibody excess Adjuvants: Freunds Complete or Incomplete adjuvant Carriers: keyhole limpet hemocyanin (KLH), bovine serum albumin (BSa), ovalbumin (oVa) and purified protein derivative of tuberculin (PPd) Monoclonal Antibodies B-lymphocytes are isolated from the spleen and fused with an immortal cell line the fused and immortalized cell line is called a hybridoma. ascites fluid has a very high concentration of antibody compared to tissue culture supernatant; Fluorophore: flurochrome, fluorescent tag/dye Specimen type Archived specimens Paraffin blocks Must heat and process through xylenes and alcohols ruins some antigens

Steps Fixation Sectioning

aldehydes

microtome Deparaffinization xylene Decreasing concentration of alcohol (100,95,70) Rehydration process Antigen Retrieval HIER 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 Detergents Triton X Tween Blocking Avoids non-specific reaction ACTIVITY block Inhibits endogenous substances that mimics reporter activity (enzyme system) SERUM block Inhibits binding of Primary IgG on the slide surface Visualization Processes Fresh Frozen Section/ Snap Frozen Tissues OCT optimal cutting temperature compound Steps Sectioning Fixation acetone or methanol or aldehydes

 Improving Antibody penetration Blocking visualization Tissue/Cell culture Steps Fixation acetone or methanol or aldehydes Improving Antibody penetration Blocking visualization Processes

Direct Labels Primary antibody conjugated with a fluorophore. Toxin with high affinity to molecular target conjugated with a fluorophore (phalloidin against f-actin) Nucleic acid probes DAPI(4',6-diamidino-2-phenylindole) Hoechst Indirect labeling

Indirect labelling with signal amplification: Strept(avidin)/Biotin

o Streptomyces avidinii. extraordinarily high affinity for biotin (also known as vitamin B7) o one of the strongest non-covalent interactions known in nature. o it is not amenable to multiplex experiments o endogenous biotin can cause background and specificity issues when performing assays with certain biotin-rich tissues and extracts (i.e., brain, liver, milk, eggs, corn) o amplify the original protien signal to improve detection of proteins expressed at low levels by forming large avidin-biotin complexes Reporter Systems Fluorophore Limitations Photobleaching: photochemical destruction of a fluorophore due to the generation of reactive oxygen species in the specimen as a byproduct of fluorescence excitation Autofluorescence:

 Flavin coenzymes (FAD and FMN: absorption, 450 nm; emission, 515 nm) and reduced pyridine nucleotides (NADH: absorption, 340 nm; emission, 460 nm) Fixation with aldehydes, particularly glutaraldehyde, can result in high levels of autofluorescence Fluorescence Overlap or bleed through Enzymes: Horseradish Peroxidase isolated from the root of the horseradish plant (Cochlearia armoracia) Activity block: quenches endogenous peroxidase by incubating with substrate (hydrogen peroxide) Calf Intestine Alkaline Phosphatase removes (by hydrolysis) and transfers phosphate groups from organic esters by breaking the P 0 bond lack of interference posed by endogenous peroxidase activity. endogenous alkaline phosphatase activity from bone, kidney, liver and some white cells can be inhibited by the addition of 1 mM levamisole to the substrate solution Substrates/Chromogen DAB 3,3 diaminobenzidinetrahydrochloride AEC 3 amino-9-ethylcarbazole Controls Normal: normal tissue Positive: tissue with known presence of the target molecule Negative negative reagent control: omitting the primary antibody on a parallel tissue section that otherwise is treated identically Non-immune IgG/serum from same species of primary Ab negative tissue or cell control Internal: normal cells present in the diseased tissue (ex. Blood vessels in tumor) Counter staining Provides visual enhancement Applications Diagnostics Pathology Research Developmental Biology Cancer Biology