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Detection of Metastatic Tumor Cells in Bone Marrow
Published in Adrian P. Gee, BONE MARROW PROCESSING and PURGING, 2020
The immunoperoxidase staining method involves a cytocentrifuge preparation of bone marrow cells where visualization of bound antibody is ultimately achieved by a chemical reaction between an enzyme and coloring agent (e.g., peroxidase with diaminobenzidene; Figure 1). This is a labor-intensive method, but has very few false positives because positive staining can be verified by light microscopic evaluation for malignant morphology. Incubation with the primary antibody can be performed when cells are either in suspension or on the coverslip. The avidin biotin method appears to be the most sensitive because of amplification of the antigen antibody binding. The sensitivity of detection varies from 1 tumor cell among 10,000 normal cells down to 1 tumor cell among 250,000 cells.13,14,21,25
Immunocytochemical Detection Systems
Published in Lars-Inge Larsson, Immunocytochemistry: Theory and Practice, 2020
Peroxidase conjugates have had numerous applications at the light and electron microscopic level.86 In studies comparing immunoperoxidase with immunofluorescence, several studies (such as the one by Brown et al.35) have noted many advantages of immunoperoxidase procedures, including availability of permanent preparations, lack of fading of the reaction product, and elimination of autofluorescence and, sometimes, of unspecific reactions. Above all, these methods were also suitable for EM immunocytochemistry (cf. Reference 86),and attempts to extend them even further to scanning electron microscopy were made.245,349
Pregnancy-Related Proteins Detected by Immunochemical or Physicochemical Methods
Published in Gábor N. Than, Hans Bohn, Dénes G. Szabó, Advances in Pregnancy-Related Protein Research, 2020
The average content of PP10 in one human term placenta amounts to 20 mg.89 The location of PP10 in human placentas was investigated by Inaba et al.88 by use of an enzyme-immunoperoxidase technique. PP10 was found to be mainly located in the syncytiotrophoblast. A strong staining was also observed in histiocytes occurring in amniotic, villous, and decidual tissues. In addition, it was found that granulocytes in normal blood also stained strongly positive for PP10; this indicated that PP10 is not specific to the placenta. Inaba et al.75 also investigated the occurrence of PP10 in malignant nontrophoblastic tumor tissues. In all, 40 out of 72 tissues (55%) showed cytoplasmic staining for PP10. Localization studies of PP10 in trophoblastic tumor tissues have been performed by Wahlstrom et al.91 PP10 was only demonstrable in the syncytiotrophoblasts of normal placentas and hydatidiform moles but not in destructive moles or choriocarcinomas.
CaMKII may regulate renal tubular epithelial cell apoptosis through YAP/NFAT2 in acute kidney injury mice
Published in Renal Failure, 2023
Zongshun Huang, Yonghua Peng, Guibao Ke, Yun Xiao, Yaqi Chen
Paraffin-embedded kidney tissue sections were deparaffinized with dimethylbenzene and hydrated with ethanol and water. The sections were placed in 0.01 M citrate buffer and heated for 20 min at 95 °C in a water bath. After cooling to room temperature, slides were immersed in 3% H2O2 to quench endogenous peroxidase activity. Slides were then washed with PBS and incubated with rabbit anti-phospho-CaMKII (Cell Signaling Technology; 1:100), rabbit anti-YAP (Cell Signaling Technology; 1:100), and rabbit anti-NFAT2 (Abcam, USA; 1:100) overnight at 4 °C. After washing with PBS, the slides were incubated for 1 h at room temperature with a horseradish peroxidase-labeled secondary antibody and then washed. Immunoperoxidase staining was performed using a Ready-to-use Immunohistochemical SP Kit (Nanjing KeyGEN Biotech, China) according to the manufacturer’s instructions. The slides were counterstained with hematoxylin and mounted before examination by light microscopy. We selected 18 random sites from 6 sections in each group to conduct quantitative analysis via Image-Pro Plus 6.0 [16,21], (Media Cybernetics, Georgia Avenue, MD, USA). Two investigators who were blinded to the original samples analyzed all images.
Reduced CXCR4 expression in associated with extramedullary and predicts poor survival in newly diagnosed multiple myeloma
Published in Expert Review of Hematology, 2022
Dangui Chen, Yang Zhan, Hong Yan, Hong Liang, Fusheng Yao, Haitao Xu
An indirect immunoperoxidase method was used. Formalin-fixed and paraffin-embedded sections were utilized for IHC with antibody [15]. During heated antigen retrieval the slides were immersed in an ethylene diamine tetraacetic acid (EDTA) buffer (potential of hydrogen, pH 8.0) and heated for 2 min in a steamer. The sections were incubated overnight at 4°C with primary antibodies specific for anti-CXCR4 antibody (1:100; human, ab124824, Abcam, Shanghai, China). The secondary antibody was goat anti-rabbit IgG H&L (HRP) (1:500; cat, ab96899, Abcam, Cambridge, United Kingdom). The sections were finally counterstained with hematoxylin, then washed and mounted in an aqueous mounting medium. Negative controls were processed in the same manner except that the primary antibody was replaced with phosphate buffer saline (PBS). The sections were observed using an BX53 fluorescence microscope(Olympus, Tokyo, Japan) and the images were analyzed (blue: nucleus, brown: target protein). IHC was evaluated by 2 experienced hematopathologists using a multihead microscope. Then the staining intensity of CXCR4 in the slides was detected. Without prior knowledge about the patients’ outcomes, the two pathologists independently graded the immunostaining intensity as follows: no or low in < 30% of myeloma cells (CXCR4-negative), and strong in ≥ 30% myeloma cells (CXCR4-positive) (Figure 1).
Combined Use of Magnesium Sulfate and Fingolimod for Antenatal Neuroprotection against Inflammation-Mediated Experimental Preterm Brain Injury in a Rat Model
Published in Fetal and Pediatric Pathology, 2022
Serenat Eris Yalcin, Mekin Sezik, And Yavuz, Mehtap Savran, Halil Asci, Ozlem Ozmen
For immunohistochemistry, three sections of each paraffin-embedded fetal brain tissues were stained with S100β, interleukin-6 (IL-6), and interleukin-10 (IL-10) antibodies. The primary and secondary antibodies were obtained from Abcam (Cambridge, UK). As primary antibodies, S100 β (anti-S100 beta antibody, Astrocyte Marker, ab868, 1/100 dilution), IL-6 (anti-IL6 antibody, ab9324, 1/100 dilution), and IL-10 (anti-IL10 antibody, ab34843, 1/100 dilution) were used. As a secondary antibody, the Mouse and Rabbit Specific HRP Plus (ABC) Detection IHC Kit (ab80436) was used. For the immunoperoxidase method, the streptavidin–biotin complex peroxidase method was used. The method was performed according to the manufacturer’s instructions, and 3,3′-diaminobenzidine (DAB) was used as the chromogen. For counterstaining, Harris hematoxylin was used, and coverslip-covered preparations were examined under light microscopy.