Symptom flowcharts and testing guidelines
Sarah Bekaert, Alison White in Integrated Contraceptive and Sexual Healthcare, 2018
Following solvent treatment, only Gram-positive cells remain stained, possibly because of their thick cell wall, which is not permeable to solvent. After the staining procedure, cells are treated with a counterstain, e.g. a red acidic dye such as safranin or acid fuchsin, in order to make Gram-negative (decolourised) cells visible. Counterstained Gram-negative cells appear red, and Gram-positive cells remain blue. Although the cell walls of Gram-negative and Gram-positive bacteria are similar in chemical composition, the cell wall of Gram-negative bacteria has a thin layer between an outer lipid-containing cell envelope and the inner cell membrane. This means that within the staining process, the cell wall loses the crystal violet colour during the use of the alcohol in the decolourisation process and takes on the red stain at the end part of the staining process. The Gram-positive cell wall is much thicker, and retains the crystal violet stain even through the decolourisation process.
Context-Driven Image Analysis: Cognition Network Language
Gerd Binnig, Ralf Huss, Günter Schmidt in Tissue Phenomics, 2018
H&E staining is, on one hand, not very specific and, therefore, solving the H&E problem might appear meaningless to many people working in digital pathology. On the other hand, H&E staining is most often used by pathologists, as it delivers valuable information. A wide range of features can be extracted, and immune cells (not the states they are in) are distinguishable from other cells. Furthermore, hematoxylin is nearly always used as a counterstain in IHC when specific proteins such as CD8 (to mark T cells) are stained. The counterstain helps to understand the scenario as a whole. It is useful for a more holistic investigation of tissue slides. It is, for example, valuable for determining ratios of numbers of specific cell or cells in a specific state stained by an antibody to all other cells stained by H. As the H counterstain visualizes nearly all types of nuclei, this is very meaningful. The specifically stained proteins in IHC are less difficult to detect, as their contrast is strong. Therefore, solving the H&E problem solves also the most difficult part of IHC slide analysis!
Enterocytozoon
Dongyou Liu in Handbook of Foodborne Diseases, 2018
The two most commonly used staining techniques include the following: (1) modified trichrome stain—developed in 1992 by Weber,83 which makes use of chromotrope 2R. It is based on Wheatley's modification of the Gomori's stain with two important modifications, using chromotrope 2R at 10× concentration and increasing the staining period. These steps not only expedited the staining process but also provided a good contrast against the background. The Weber's trichrome stain stains the spore of E. bieneusi reddish-pink in color, which appear as rosy oval bodies with a nearly colorless posterior vacuole. The polar filament may take up stronger stain giving a darker contrast at the equator of the spore. The background takes the color of the counterstain used, malachite green, Fast green or aniline blue may be used for the same.2,79,82
Evaluation of sodium orthovanadate as a radioprotective agent under total-body irradiation and partial-body irradiation conditions in mice
Published in International Journal of Radiation Biology, 2021
Yuichi Nishiyama, Akinori Morita, Bing Wang, Takuma Sakai, Dwi Ramadhani, Hidetoshi Satoh, Kaoru Tanaka, Megumi Sasatani, Shintaro Ochi, Masahide Tominaga, Hitoshi Ikushima, Junji Ueno, Mitsuru Nenoi, Shin Aoki
At 4 and 7 days post-irradiation, mice were intraperitoneally injected with 5-Bromo-2′-deoxyuridine (BrdU; 50 mg/kg; Sigma) 1 h prior to euthanasia. The excised ileum was washed with 10 mM phosphate-buffered saline, fixed in 10% formalin, and embedded in paraffin. Cross sections of intestinal tract (5 μm thick) were stained with hematoxylin-eosin (HE) to examine morphological change of intestinal epithelium. Cells incorporating BrdU in the intestinal crypts were detected immunohistochemically using the BrdU in situ detection kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer's instructions. Hematoxylin was used as counterstain. Intestinal tissue images were photographed with a BZ-9000 microscope (Keyence, Osaka, Japan), and three observers counted a surviving crypt having 5 or more BrdU-positive cells using a BZ-X analyzer software (Keyence) (Saha et al. 2011). The percentage of surviving crypts relative to the mean crypt number in mice that had been treated with NS only was calculated from a minimum of 3 cross-sections in each mouse.
Identification of possible Lynch syndrome in endometrial carcinomas at a public hospital in South Africa
Published in Southern African Journal of Gynaecological Oncology, 2020
R Wadee, W Grayson
The four mismatch repair antibodies, namely MLH1 (Novocastra, UK), PMS2 (Novocastra, UK), MSH2 (Novocastra, UK) and MSH6 (Novocastra, UK), were used to stain 4 μm deparaffinised sections. A microtome was used to cut tissue sections from paraffin-embedded blocks. The tissue sections were then floated onto slides, which subsequently underwent drying overnight at 60°C. These four immunohistochemical stains had previously been optimised according to departmental standard operating procedure. Staining of tissue sections includes the treatment of primary antibody serum. Each of the tissue sections then underwent staining with the following antibodies: MLH1 (Clone ES05, 1:50), PMS2 (Clone MOR4G, 1:50), MSH2 (Clone 25D12, 1:50) and MSH6 (PU29, 1:50). A mouse linker resulted in increased expression of antigens. The sections were washed with Tris buffered saline (TBS) at pH 7.6. Immunohistochemistry was undertaken using an automated staining machine (DAKO Autostainer Link 48, Denmark). A ready-made solution, the EnVision™ FLEX target Retrieval Solution, High pH, was used for antigen retrieval. The chromogen used was 3,3′ diaminobenzidine hydrochloride solution (DAB, Sigma, USA), which produced a brown pigment. Meyer's haematoxylin was the counterstain used on tissue sections. Appropriate positive and negative control tissue sections were used.
3D volume segmentation and reconstruction. Supervised image classification and automated quantification of superparamagnetic iron oxide nanoparticles in histology slides for safety assessment
Published in Nanotoxicology, 2021
Anna Bogdanska, Oliviero L. Gobbo, Yuri Volkov, Adriele Prina-Mello
Following the SPION biodistribution and pharmacokinetic study (Gobbo et al. 2015), the organs were fixed in 4% formalin and paraffin embedded. Samples from two animals per time point were used in this study. Four consecutive sections (5 µm thick) were collected at three levels. First section was stained with hematoxylin and eosin (H&E) stain for general toxicity studies (data not shown). The following two sections were stained with Perls’ Prussian blue (PPB) staining as detailed in previous publication with minor modifications (Edge et al. 2016a). Briefly, rehydrated sections were covered with equal parts of hydrochloric acid (HCL) and potassium ferrocyanide (K4[Fe(CN)6]·3H2O) for 20 min. Slides were washed in distilled water and counterstained with eosin for 5 min. Following incubation periods, slides were rinsed with distilled water before being dehydrated in a graded series of alcohol rinses. Slides were then cleared in two baths of xylene, sealed under coverslips with DPX mounting medium and examined under light microscope. To evaluate if any iron-positive staining is masked by the counterstain, third section was stained with PPB stain omitting eosin step in the outlined protocol (data not shown).
Related Knowledge Centers
- Crystal Violet
- Haematoxylin
- Malachite Green
- Microscope
- Eosin
- Staining
- Fuchsine
- Gimenez Stain
- H&E Stain
- Gram Stain