Basics of Immunohistochemistry

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Basics of Immunohistochemistry Vivien Schacht1 and Johannes S. Kern2 1Department of Dermatology, Hannover Medical School, Hannover, Germany; schacht.vivien@mh-hannover.de 2Department of Dermatology, Medical Center – University of Freiburg, Freiburg, Germany; johannes.steffen.kern@uniklinik-freiburg.de

INTRODUCTION Immunohistochemistry (IHC) : Demonstrates specific antigens in formalin-fixed, paraffin-embedded (FFPE) tissues. Antigen-antibody construct is visualized through light microscopy => morphology of the tissue around the specific antigen is visible. Results are reported semiquantitatively (0;+;++;+++) and have important diagnostic and prognostic implications.

HISTORY 1940 immunofluorescence staining on frozen sections based on antigen-antibody interaction was presented by Coons. 1974 Taylor and Burns developed IHC on routinely processed FFPE tissues. 1975 Köhler and Milstein presented the hybridoma technique to produce monoclonal antibodies. => broad range of antigens and high staining quality

How Is Immunohistochemistry Performed? Tissue processing and epitope retrieval Formalin fixation Cutting sections Mount on glass slides Enzyme digestion Antigen-antibody interaction Direct or indirect method Visualization through different detection systems Fluorescent compounds or active enzymes

Quality Control Purpose: Sensitive and specific, reproducible and standardized Positive control: Sample containing the antigen of interest Negative control: Same sample as for the positive control, but primary antibody replaced by non-binding immunoglobulin Validation of IHC: Round robin tests Staining various tissue and tumor types Comparing staining results of different antibodies that recognize similar proteins.

Immunohistochemical Techniques Direct method Indirect method with polymer chain detection system

in Human Melanocytic Tumors p16INK4a Expression in Human Melanocytic Tumors Scurr et al. 2011

Immunohistochemical Results for Select BRAF V600E Mutation Positive Cases by DNA Analyses Feller et al. 2013