An Introduction to the Performance of Immunohistochemistry

Abstract

Summary Immunohistochemistry (IHC) is a powerful technique that exploits the specific binding between an antibody and antigen to detect and localize specific antigens in cells and tissue, most commonly detected and examined with the light microscope. A standard tool in many fields in the research setting, IHC has become an essential ancillary technique in clinical diagnostics in anatomic pathology (1) with the advent of antigen retrieval methods allowing it to be performed conveniently on formalin fixed paraffin embedded (FFPE) tissue (2, 3) and automated methods for high volume processing with reproducibility (4). IHC is frequently utilized to assist in the classification of neoplasms, determination of a metastatic tumor’s site of origin and detection of tiny foci of tumor cells inconspicuous on routine hematoxylin and eosin (H&E) staining. Furthermore, it is increasingly being used to provide predictive and prognostic information, such as in testing for HER2 amplification in breast cancer (5) in addition to serving as markers for molecular alterations in neoplasms, including IDH1 and ATRX mutations in brain tumors (6). In this section we describe the basic methods of immunohistochemical staining which has become an essential tool in the daily practice of anatomic pathology worldwide.

Tissue Expression of Melanoma-associated Antigen A6 and Clinical Characteristics of Gastric Cancer

“…However, to translate our results into clinical practice, MAGEA6 expression must be shown to be effective for monitoring postoperative disease and for selecting an optimal multimodal management strategy. Another important finding of the current study is that in situ MAGEA6 expression was detected using IHC, which is a standard clinical technique that can readily be applied to archived formalin-fixed paraffin-embedded samples (29)(30)(31). Our results indicate that IHC assays for MAGEA6 expression may enable physicians to stratify patients at risk of postoperative recurrence at the time of endoscopic biopsy or surgical resection of the primary GC.…”

Immunofluorescence Staining of Paraffin Sections Step by Step

“…Dewaxing steps have been described slightly differently by other protocols e.g., incubating the paraffin sections in xylene I-III for 5-15 min each, skipping the incubation in xylene/ethanol or adding a step of 50% ethanol for 5 min (Gandhi and Khare, 2018;Magaki et al, 2019). In our lab, we never obtained altered outcomes after changing these steps.…”

“…Western blot, blocking peptides, and negative controls are the commonly used validation methods (Skliris et al, 2009;Bordeaux et al, 2010). In order to confirm the antibody's specificity, it is always recommended to use positive and negative controls based on experimental settings and lab facilities (for reviews, see Hsi, 2001;Shi et al, 2007;Saper, 2009;Bordeaux et al, 2010;Holmseth et al, 2012;Magaki et al, 2019). (f) The storage conditions and the expected expiry dates of the antibodies, as usually stated in the accompanying antibody sheets, should be cross-checked and noted.…”

“…Primary antibody solutions are prepared on the first day of the experiment during the blocking step (section "Dewaxing Step"), while the secondary antibody solutions are prepared on the second day during the second rinsing step (section "Second Rinsing Step"). For the first trial, preferably three different concentrations of a primary antibody should be tested, and these should include the dilution recommended by the producer or in the literature as well as concentrations above and below the recommended dilution (Magaki et al, 2019). For example, if a dilution of 1:300 is recommended, a scientist could test 1:100, 1:300, and 1:500.…”

Biomarkers of resistance to immune checkpoint inhibitors in non-small-cell lung cancer: myth or reality?

“…The standardization of pre-analytic variables is also required [80] , as the definition of precise thresholds varies.…”