Laboratory Procedures and Management
Jeremy R. Jass in Understanding Pathology, 2020
The next stage involves removing the wax and staining the sections. The slides are loaded into racks and placed in xylene to dissolve the wax. This is followed by 100%, 90% and 70% alcohol and finally back to water, whereby the sections become completely transparent. In order to be studied by microscopic examination, they are dyed with chemicals, usually haematoxylin, which stains nuclei blue (due to the presence of acidic DNA) and eosin which imparts a pink colour to the cytoplasm (H&E sections). The sections are then dehydrated and returned to xylene before being covered with a glass cover slip with the aid of runny and transparent mounting medium that slowly hardens. A senior technologist checks the final quality of the slides and then they are passed to the anatomical pathologist for reporting.
Image Analysis for Tissue Phenomics
Gerd Binnig, Ralf Huss, Günter Schmidt in Tissue Phenomics, 2018
Ideal staining intensity for image analysis studies is often lower than for manual assessments, as maximizing dynamic range is more critical than highly visible staining; image analysis is not subject to the detection limits of the human eye, and intense staining conditions designed for binary pathology decisions can obscure information from the high and low ends of the staining intensity spectrum. Minimization of background staining is another reason to forego very intense stains, which can make accurate segmentation of individual cells in darkly stained areas problematic. Hematoxylin can be just as important to optimize as the stain itself, as nuclear morphologies and hematoxylin intensity can be invaluable tools in classifying both biomarker-positive and biomarker-negative cells into different cell types, which can then be aggregated into regional definitions. While CNT-based tissue phenomics image analysis approaches are not tied to nuclear objects as the starting point of an analysis, many simpler tools rely upon them for cell detection, meaning in these cases also optimized counterstains will give the best possible results from image analysis experiments. Tissue information made inaccessible through poor histology cannot be recovered by any image analysis system. Scanning of tissues is the final step in making tissue ready for image analysis and should be performed by experienced personnel on a high-quality scanner. Scans should be in focus for the entire slide if possible, and at a minimum, the regions of interest must be consistent and analyzable.
The Medicolegal Autopsy
Kevin L. Erskine, Erica J. Armstrong in Water-Related Death Investigation, 2021
All major organs and glands are individually sectioned multiple times using a scalpel to look for structural abnormalities, disease, and injury. A small tissue sample of each major organ is taken and submitted in a fixative solution prior to histological preparation and later examined under the microscope by the forensic pathologist (Figure 7.51). This histological preparation involves standard application of stains called hematoxylin and eosin to each section of tissue in order to bring out the unique characteristics of the tissue and cellular structure (Figure 7.52). The number of samples taken for any given case is dictated by the complexity of the autopsy and the need to adequately characterize the extent of injury or disease. A typical autopsy may include sampling of the heart, lungs, liver, kidneys, and brain, more or less, depending on the case. In certain cases, the pathologist may opt not to submit tissue samples for microscopic examination. Sampling with long-term retention of small portions of tissue in a stock jar containing formalin fixative is routine. These reserved tissues are kept for a pre-defined and often limited time period according to the protocol of the office and is done in the event a future need for a microscopic examination of those tissues arises.
Fe3O4 Nanopowders: Genomic and Apoptotic Evaluations on A549 Lung Adenocarcinoma Cell Line
Published in Nutrition and Cancer, 2020
Ayse Kaplan, Hatice Mehtap Kutlu, Gulsen Akalin Ciftci
The amount of apoptosis was observed by hematoxylin and eosin (H&E), TUNEL paraffin embedded, BrdU, Bcl-2 and Bax staining using light microscopy (Fig. 3). Hematoxylin is a blue-violet substances, which dye nucleic acids. Eosin is pink substance, which dye proteins. While core is stained in blue, the cytoplasm and the extracellular matrix are stained in pink (19). The DNA strand breaks are determined using terminal deoxynucleotidyl transferase [TdT] - mediated deoxyuridine triphosphate [dUTP] cutting and labeling (TUNEL) (20). The Bcl-2 is anti-apoptotic protein and labelled by an antibody for expression level at apoptosis. The overexpressed bcl-2 blocks apoptosis. The bax disables bcl-2 gene and thus trigger apoptosis (21). Therefore, we calculated the apoptosis index of A549 cells by immunocytochemical staining. As a result of immunocytochemical staining (TUNEL, BrdU, Bcl-2, and Bax), the cisplatin induced apoptosis compared to control cells (Fig. 3a and b) (Table 5). However, it has been shown that the iron oxide nanopowders triggered apoptosis at TUNEL, BrdU, Bcl-2 staining (Fig. 3a and c) (Table 5).
In vivo percutaneous permeation of gold nanomaterials in consumer cosmetics: implication in dermal safety assessment of consumer nanoproducts
Published in Nanotoxicology, 2021
Mingjing Cao, Bai Li, Mengyu Guo, Ying Liu, Lili Zhang, Yaling Wang, Bin Hu, Jiayang Li, Duncan S. Sutherland, Liming Wang, Chunying Chen
HE staining is a popular and established method in histology. Herein it was used to evaluate the dermal lesions caused by exposure of cosmetic creams and extracted Au nanosheets. 4 μm serial paraffin sections sliced carefully from skin surface to subcutaneous layer were deparaffinized, rehydrated, and stained with hematoxylin and eosin. For IHC analysis, 4 μm serial sections were firstly deparaffinized and rehydrated, followed by retrieving the masked antigens with 0.01 M sodium citrate buffer in the microwave (96 °C for 10 min). After blocking endogenous peroxidase with 30% H2O2, the sections were incubated with primary antibodies overnight at 4 °C and followed by the incubation with secondary antibodies labeled by horseradish peroxidase (HRP) at 37 °C for 1 h. Hematoxylin was used for nuclei staining.
The lethal effects and determinants of microcystin-LR on heart: a mini review
Published in Toxin Reviews, 2021
Muwaffak Alosman, Linghui Cao, Isaac Yaw Massey, Fei Yang
Researchers have used HE stains for more than a century and they are still effective in recognizing a wide range of tissue types and changes in morphology that forms the basis of a cancer diagnosis (He et al. 2014, Iseki et al. 2018). The stain remains unchanged for many years since it is effective with several fixatives and shows a wide range of nuclear, cytoplasmic, and extracellular matrix features. Hematoxylin contains a deep blue color that stains nucleic acids. Eosin, on the other hand, is pink in color and spans proteins nonspecifically. While a typical tissue nucleus has a blue stain, the extracellular matrix and cytoplasm are stained pink but in varying degrees (Rode and Eisel 2004). In the presence of abundant polyribosomes, the cytoplasm is likely to show a distinct blue cast. Additionally, the lack of staining in an area next to the nucleus helps in identifying the Golgi zone (Fischer et al. 2008). This implies that the stain uncovers structural information. Nonetheless, one of the shortcomings of hematoxylin staining is that it is not compatible with immunofluorescence. This notwithstanding, the method is effective in staining one serial paraffin section from an organ or tissue where immunofluorescence is performed. Generally, hematoxylin without eosin helps in counterstaining for numerous hybridizations or immunohistochemical procedures that need colorimetric substrates.
Related Knowledge Centers
- Cell Biology
- Chemical Compound
- Counterstain
- Histology
- Eosin
- Staining
- H&E Stain
- Papanicolaou Stain
- Redox
- Hematein