ABSTRACT
CONTEXT AND OBJECTIVE: The conversion of immunohistochemical (IHC) results from 3-dimensional tissue to a 2-dimensional visual image without considering tissue thickness poses a considerable risk of misleading IHC intensities. The present study aimed to clarify whether tissue thickness interferes with the estimation of IHC staining intensity and to introduce a control system to manage it. DESIGN: We prepared cell lines that are used as controls for human epidermal growth factor receptor 2 (HER2) IHC (MDA-MB-231, MDA-MB-175VII, MDA-MV-453, and SK-BR-3), a polyclonal antibody for HER2, an interferometry to measure the tissue thickness of formalin-fixed paraffin-embedded sections, a microscope with a Halogen or an LED light source, a complementary metal-oxide semiconductor camera in which the output signal can be corrected to γ=1, and a program to estimate color elements (hue, saturation, and luminance). It was examined whether tissue thickness interferes with the experimental scoring systems and practical classification of the routine HER2 scoring system. RESULTS: A noncellular control was shown to be better than a cellular control for managing tissue thickness. The IHC intensity for HER2 was correlated with tissue thickness (R2=0.8094), even under the less-standardized condition, but this correlation was better under the improved standardized condition using corrected γ=1 (R2=0.9282). Discrepancies in practical HER2 scores were increased in sections with thicknesses <2 and >5 µm. A control system to manage tissue thickness was introduced. CONCLUSIONS: Tissue thickness interferes with the estimation of the IHC intensity of HER2 in both experimental and practical scoring systems. A control system for managing tissue thickness is essential to increase the benefits of IHC as a standardized assay for clinical applications.
Subject(s)
Biomarkers, Tumor/metabolism , Breast Neoplasms , Immunohistochemistry , Receptor, ErbB-2/metabolism , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Female , Humans , Microscopy , Paraffin EmbeddingABSTRACT
Temperature-responsive culture surfaces make it possible to harvest transplantable carrier-free cell sheets. Here, we applied temperature-responsive polymer for polycarbonate surfaces with previously developed closed culture devices for an automated culture system in order to fabricate transplantable stratified epithelial cell sheets. Histological and immunohistochemical analyses and colony-forming assays revealed that corneal epithelial and oral mucosal epithelial cell sheets could be harvested with the temperature-responsive closed culture devices. The results were similar to those obtained using temperature-responsive culture inserts. These results indicate that the novel temperature-responsive closed culture device is useful for fabricating transplantable stratified epithelial cell sheets.
Subject(s)
Cornea/pathology , Epithelial Cells/cytology , Mouth Mucosa/pathology , Tissue Engineering , 3T3 Cells , Animals , Automation , Cell Culture Techniques/methods , Culture Media , Immunohistochemistry , Mice , Microscopy, Phase-Contrast , Polycarboxylate Cement/chemistry , Polymers/chemistry , Porosity , Rabbits , Stem Cells , TemperatureABSTRACT
We defined the staining applicability of Histofine MOUSESTAIN, Mouse MAX-PO and Rat MAX-PO for fixed frozen and fresh frozen tissue sections besides paraffin embedded tissue sections. Two different types of cool air dried tissue sections, fresh frozen tissues and fixed frozen tissues of nine-week old Slc: ICR mouse and nine-week old Slc: SD rat, were prepared for immunohistochemistry (IHC) staining in accordance with conventional method. Additional two steps of 1 and 2 to our recommended procedure for paraffin embedded tissue sections were examined as follows: STEP 1: Adjust reaction time of polymer; STEP 2: Add 0.2% of glutaraldehyde (GA) solution before the STEP 1, when background staining is still observed by STEP 1. The steps 1 and 2 resulted in elimination of background staining of most Histofine series. Therefore, polymer detection systems, Histofine series, are applicable for IHC staining of two different types of frozen tissue sections as well as paraffin embedded tissue sections.
Subject(s)
Frozen Sections/methods , Immunohistochemistry/methods , Paraffin Embedding/methods , Staining and Labeling/methods , Animals , Glutaral , Humans , Male , Mice , Mice, Inbred ICR , Polymers , Rats , Rats, Sprague-DawleyABSTRACT
It is critical to evaluate the overexpression of human epidermal growth factor receptor 2 (HER2) adequately in breast cancer cases. The aim of the present study is to assess the characteristics and usefulness of a novel monoclonal antibody, clone SV2-61gamma. The overexpression of HER2 was studied on paraffin sections of 50 breast cancers using a polyclonal antibody assay (HercepTest) and monoclonal antibodies (clones SV2-61gamma and CB11). Gene amplification of HER2 was determined by fluorescent in situ hybridization (FISH) using a HER2/CEP17DNA probe kit. Fifty tumors were scored as 0 (50.0%), 1+ (26.0%), 2+ (4.0%), or 3+ (20.0%) by immunohistochemistry using a monoclonal antibody, clone SV2-61gamma. The specificities of immunohistochemistry were 72.0% for the polyclonal antibody, 97.0% for SV2-61gamma, and 92.0% for CB11. The concordances with FISH were 78.0% for the polyclonal antibody, 98.0% for SV2-61gamma, and 94.0% for CB11. The positive predictive value of SV2-61gamma (92.0%) was higher than those of the polyclonal antibody (50.0%) and CB11 (79.0%). A monoclonal antibody clone SV2-61gamma showed very specific staining, with the best concordance with FISH, and it is useful for the evaluation of HER2 overexpression.
