Morphologic Detection of Multiple Steroid Binding Sites in Breast Cancer by Histochemistry and By Immunocytochemistry with Monoclonal Antireceptor Antibody
P. Pertschuk Louis, Lee Sin Hang in Localization of Putative Steroid Receptors, 2018
ERICA was performed in the following way according to the technique developed by G. Greene and W. King, University of Chicago. Frozen tissue sections were fixed in picric acid/paraformaldehyde, exposed to ovalbumin to reduce nonspecific binding, and then incubated with 40 to 80 (x€ of pooled monoclonal antiestrophilin antibody per milliliter PBS. We used the D547 Sp-y and D75Sp-y antibodies of Dr. Greene. After exposure to the pool for 30 min, the sections were washed in PBS, reacted with goat anti-rat IgG (since the monoclonal antibody was of rat origin), and then with rat peroxidase antiperoxidase complex (PAP). The reaction product was made visible with diaminobenzidine tetrahydrochloride and H2O2 in the usual way. No counterstain was employed. For control sections, normal rat IgG was substituted for the monoclonal antibody pool, or the pool was eliminated entirely. In some experiments, specifically absorbed antibody, supplied by Dr. Greene, was used. Specimens were considered positive for ER when at least 10% of the component tumor cells exhibited staining. Cases (103) previously analyzed with 6-FE, 17-FE, and DCC were studied.
The Type II Pneumocyte
Jacques R. Bourbon in Pulmonary Surfactant: Biochemical, Functional, Regulatory, and Clinical Concepts, 2019
They also contain various amounts of peroxisomes. These structures constitute a separate class of cytoplasmic organelles that are best characterized by their enzyme content: at least one hydrogen peroxide-generating enzyme and large amounts of H2O2-destroying catalase.66 Following diaminobenzidine treatment, they appear as membrane-bound bodies containing an electron-dense reaction product. Their presence in mouse and rat lung tissues was first demonstrated by Petrik in 197166 within type II pneumocytes and Clara cells. Since then, microperoxisomes have been evidenced in type II cells of a number of mammalian species such as hamster, guinea pig, monkey,67 and pig.68 In addition, they have recently been identified in the lipid-filled interstitial cells of postnatal rat lung.69
Immunochemical Approaches to the Diagnosis of Alzheimer Disease
Robert E. Becker, Ezio Giacobini in Alzheimer Disease, 2020
The methods of detection utilized in these assays fall into three categories: radioactivity, enzyme amplification and, more recently, scintillation proximity detection. Radioactivity can be counted directly. The scintillation proximity assay can also be counted directly. A conformationally sensitive ligand is attached to the antibody. Binding of the antigen alters the antibody resulting in the ligand giving off photons which can be detected by a scintillation solution. Alternatively, a functional enzyme, such as peroxidase or ß-galactosidase, can be attached to the antibody. Incubation of the enzyme with a particular substrate produces a colored reaction product that can be visualized; for instance incubation of peroxidase with 2,2-diaminobenzidine produces a brown reaction product.
Follicle-stimulating hormone peptide-conjugated nanoparticles for targeted shRNA delivery lead to effective gro-α silencing and antitumor activity against ovarian cancer
Published in Drug Delivery, 2018
Shan-Shan Hong, Ming-Xing Zhang, Meng Zhang, Yi Yu, Jun Chen, Xiao-Yan Zhang, Cong-Jian Xu
Immunocytochemistry and immunohistochemistry were used to detect the expression of FSHR and gro-α in ovarian cancer cells and tissues. HEY human ovarian cancer cells were seeded in 24-well plates and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum. After fixation, cells were incubated with FSHR antibody (Abcam, Ltd, San Francisco, CA, USA) or gro-α antibody (Abcam Ltd, HK) at 4 °C overnight. Then, the cells were incubated with HRP-conjugated anti-rabbit IgG (Abcam Ltd, San Francisco, CA, USA) for 30 min at room temperature. The staining reaction was performed with diaminobenzidine. The cells were counter-stained with hematoxylin and imaged by light microscopy (Olympus Corporation, Tokyo, Japan). For ovarian cancer tissue, the procedures were the same as the cells, except the tissue samples were subjected to deparaffinization and rehydration before incubation with antibodies.
Tumor-infiltrating IL-17A+ cells determine favorable prognosis and adjuvant chemotherapeutic response in muscle-invasive bladder cancer
Published in OncoImmunology, 2020
Zewei Wang, Quan Zhou, Han Zeng, Hongyu Zhang, Zhaopei Liu, Qiuren Huang, Ying Xiong, Jiajun Wang, Yuan Chang, Qi Bai, Yu Xia, Yiwei Wang, Yu Zhu, Le Xu, Bo Dai, Li Liu, Jianming Guo, Jiejie Xu
Tissue microarray (TMA) construction and the immunohistochemistry protocol were conducted as previously described.17 For each patient, three sections of tumor tissue were taken from paraffin blocks to construct TMA. Primary antibodies (listed in supplementary table 2) were applied to sections at 4°C overnight for immunohistochemical staining. For different T helper cells, TH1 was defined as CD4+ T-bet+ cells and TH2 was defined as CD4+ GATA3+ cells. Then, slides were treated with polyperoxidase-conjugated IgG (OriGene). Staining was performed with diaminobenzidine solution (Biocare Medical) under a microscope and counterstained with hematoxylin. The primary antibody was omitted for the negative controls. The positive cells were enumerated from the representative view of the three sections in high-power field (HPF) and an average number was adopted. Two pathologists who were unaware of patient information evaluated the staining of each specimen with the assistance of Image-Pro Plus 6.0 (Media Cybernetics Inc.). In case of disagreement, the slides were reviewed and the two observers achieved a consensus.
Unraveling enhanced brain delivery of paliperidone-loaded lipid nanoconstructs: pharmacokinetic, behavioral, biochemical, and histological aspects
Published in Drug Delivery, 2022
Saleha Rehman, Bushra Nabi, Amaan Javed, Tahira Khan, Ashif Iqubal, Mohammad Javed Ansari, Sanjula Baboota, Javed Ali
For immunohistochemical analysis, sections of 5 μm thickness were cut, and paraffin was removed using xylene. The brain sections were rehydrated with graded ethanol series, washed with double distilled water, exposed to antigen retrieval using citrate buffer (pH 6), and lastly allowed to cool at room temperature for 10 minutes. The background staining was removed by incubating the sections in 4% hydrogen peroxide for 15 minutes. Sections were washed thrice using tris-buffered saline and incubated with primary Nrf-2 antibody (1:100) at 4 °C overnight. After that, sections were rinsed using the buffer and then incubated for 1 h with a peroxidase-conjugated secondary antibody. The reaction was visualized using diaminobenzidine solution. Fluorescence motic microscope (Motic AE31) facilitated with Fiji software was used to observe histological alterations in brain sections and for the semi-quantification of protein expression using the reciprocal intensity method. For these immunohistochemically stained hippocampi and frontal cortex slides, the percentage of positive neurons was estimated (Iqubal et al., 2019; Sharma et al., 2018).
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