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Antibodies and Antisera
Published in Lars-Inge Larsson, Immunocytochemistry: Theory and Practice, 2020
A third kind of specificity can be termed diagnostic specificity and reflects the reliability of immunocytochemistry/immunohistology for detecting certain diseases. This may involve both biochemical specificity (e.g., for tumor marker molecules and hormones), anatomical specificity (e.g., in diagnosis of blood cell diseases) or neither (e.g., autoimmune antibody screning). The criteria involved in diagnostic specificity include reproducibility, precision, false negatives and positives, etc., as in all clinical tests. Needless to say, this approach represents a major useful application of immunocytochemistry with respect to both diagnosis of cancer, and autoimmune and microbiological diseases. In the latter case, anatomical specificity, particularly of viral structures in immunoelectron microscopic applications, may enter the picture. A very special case may be referred to as induced specificity and involves the introduction of antigens to cells or tissues that do not ordinarily express these antigens. Examples include viral infection of cells in order to trace pathways for viral protein export and processing, as well as introduction of foreign genes in pro- or eukaryotes. Controls, of course, are unperturbed control cells. Only rarely will the issue of biochemical specificity enter this area of research.
Ultrastructural Immunocytochemistry
Published in Joan Gil, Models of Lung Disease, 2020
Samuel S. Spicer, Bradley A. Schulte
In a third area of application, immunocytochemistry aims to gain information about diagnosis, classification, and pathogenesis of disease processes (DeLellis et al., 1979; Spicer, 1987). Cells differ more in chemical composition than in structural features. Chemical analysis can therefore potentially show differences between normal and diseased cells that are not discernible morphologically. Despite its limited quantitative capacity, immunocytochemical examination often surpasses biochemical analysis in the ability to detect chemical characteristics or changes in specific types of diseased cells. By identifying specialized cell constituents, for example, immunoglobulin, light microscopic immunostaining has contributed to the diagnosis and classification of lymphoid neoplasms (Garvin et al., 1974; Lukes et al., 1978; Janossy et al., 1980). Immunostains represent diagnostic probes in diverse areas of pathology including tumor markers (Burns et al., 1978), cutaneous immunmopafhology (Harrist and Mihm, 1979) and renal disease (Maclver et al., 1979). Applications of ultrastructural immunostaining to lung are detailed in the final section of this chapter. The prospect for future contributions to pulmonary biology and pathology seems bright, if one extrapolates from past experience.
Eosinophils in a Guinea Pig Model of Allergic Airways Disease
Published in Gerald J. Gleich, A. Barry Kay, Eosinophils in Allergy and Inflammation, 2019
M. G. Campos, M-C. Seminario, J. K. Shute, T. C. Hunt, S. T. Holgate, M. K. Church
Only one of the monoclonal antibodies (designated 8A12) was found to be suitable for use in immunocytochemistry. This stained eosinophils strongly and specifically in cytospin preparations, frozen sections, or wax-embedded sections (after protease digestion). Either indirect immunofluorescence or alkaline phosphatase–anti-alkaline phosphatase techniques could be used to visualize the staining. This resulted in strong granular staining of the eosinophils and proved to be an extremely sensitive means of localizing this cell type in tissue sections. The 8A12 monoclonal was also capable of localizing MBP deposited extracellularly by eosinophils at sites of allergic inflammation. Staining with this antibody was specific for eosinophils and did not stain other guinea pig leukocyte types.
Differentiation and Maturation Effect of All-Trans Retinoic Acid on Cultured Fetal RPE and Stem Cell-Derived RPE Cells for Cell-Based Therapy
Published in Current Eye Research, 2022
Tingyu Yan, Na Yang, Wei Hu, Xinxin Zhang, Xuedong Li, Youjin Wang, Jun Kong
P1 and P5 fRPE cultured for 1 month were compared. Using RT-qPCR analysis, we assayed the mRNA expression levels of RPE markers including RPE65, ZO-1, E-cadherin, and mesenchymal markers including α-SMA, FN, N-cadherin. We found that expression of RPE65, E-cadherin, and ZO-1 in fRPE cells that underwent EMT changes were significantly decreased and expression of a-SMA, N-cadherin, and FN were significantly increased by 4.12-folds, 3.21-folds, and 10.26-folds, respectively (p < 0.05) (Figure 3(a)). In a parallel approach, western blotting results demonstrated that the protein expression of RPE markers was downregulated and the protein expression of EMT markers was upregulated in EMT-fRPE cells (Figure 3(b)). In addition, immunocytochemistry results strengthened the findings (Figures 3(c,d)).
RhoA Activation Decreases Phagocytosis of Trabecular Meshwork Cells
Published in Current Eye Research, 2021
Tomokazu Fujimoto, Saori Sato-Ohira, Hidenobu Tanihara, Toshihiro Inoue
TM cells were cultured on gelatin-coated glass cover slips for immunocytochemistry. Immunocytochemistry was conducted as described previously.25 The cells were fixed with 4% (v/v) paraformaldehyde in PBS for 15 min at room temperature, and then washed with cytoskeletal buffer (10 mM 2-morpholinoethansulfonic acid potassium salt, 150 mM NaCl, 5 mM EGTA, 5 mM MgCl2, and 5 mM glucose, pH 6.1). For permeabilization, cells were treated with 0.5% (v/v) Triton X-100 in PBS for 12 min at room temperature. The cells were then blocked with serum buffer (10% FBS and 0.2 mg/mL sodium azide in PBS) at 4°C for at least 2 h and were then treated with anti-vinculin, anti-YAP, or anti-TAZ antibodies at 4°C overnight. Afterwards, the cells were incubated with the anti-mouse IgG secondary antibody Alexa Fluor 488, and Alexa Fluor 546 phalloidin at room temperature for 30 min. After the cells had been mounted with VECTASHIELD mounting medium containing 4ʹ, 6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA), they were observed under an all-in-one epifluorescence microscope (BZ-X710; Keyence). We performed immunostaining in at least three independent experiments.
The Texas Society of Pathologists: molded by the legacy of pathology and focused on excellence in medicine for 100 years and beyond
Published in Baylor University Medical Center Proceedings, 2021
Pathology has always been an opportunistic and eclectic science, taking advantage of advances in basic sciences to conduct basic and translational research ultimately aimed at the elucidation of the etiology and pathogenesis of human diseases. In the 18th and 19th centuries, autopsy pathology was primarily responsible for the scientific elucidation of many human diseases. In the 20th and 21st centuries, autopsy pathology has continued to be primarily responsible for the discovery or elucidation of the pathogenesis of new diseases, such as acquired immunodeficiency syndrome due to human immunodeficiency virus, as well as documentation of effects of new therapies.138–141 Both the discovery process as well as diagnostic pathology have been enhanced by the coupling of gross examination and light microscopy with new techniques, including electron microscopy, fluorescence microscopy, histochemistry, and immunohistochemistry. From the 1970s onward, immunocytochemistry has become a powerful and ubiquitous component of diagnostic pathology.142