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Probiotics Modulate Cell Signaling Pathway and Innate Cytokine Responses to Oral HRV Vaccine in HGM-Transplanted Gn Pigs
Published in Lijuan Yuan, Vaccine Efficacy Evaluation, 2022
Ileum tissue from Gn pigs was fixed in buffered formalin, embedded in paraffin, and cut into serial sections (4 µm) (Fu et al., 2014). Deparaffinized and rehydrated sections were boiled in 10 mM sodium citrate buffer (pH = 6.0) for 10 min. After washing twice with Tris-buffered saline with Tween-20 (TBST), sections were blocked with 10% normal goat serum in TBST for 1 h at room temperature. Sections were incubated with primary IFN-γ (1:300 v/v, Cell Sciences, Canton, MA, USA), CD80 (1:200 v/v, Ancell, Bayport, MN, USA), p38, p-p38, ERK, or p-ERK antibodies (1:300 v/v, Cell Signaling Technology, Danvers, MA, USA) overnight at 4° C. After washing three times with TBST, the HRP-conjugated secondary antibody was added and the sections were incubated for 1 h at room temperature. The Diaminobenzidine-HRP detection system was added and sections were incubated at room temperature. Sections were then counterstained with hematoxylin, dehydrated, and cover-slipped. Assessment of positivity of IHC staining (van Diest et al., 1997) was conducted under an ECLIPSE Ti microscope (Nikon Corp., Tokyo, Japan).
Ultrastructural Immunocytochemistry
Published in Joan Gil, Models of Lung Disease, 2020
Samuel S. Spicer, Bradley A. Schulte
Intensification of electron opacity at a reactive site could prove advantageous for localizing antigens present in the section at low concentration or low level of immunoreactivity. Postfixation with osmium tetroxide after immunostaining is generally thought to enhance the contrast between positive sites and background. Chelating the HRP produced reaction product of diaminobenzidine with cobalt and other metals changes the color and intensity of the stain at the light microscopic microscopic level and could affect the sensitivity of the electron microscopic stain.
Immunochemical Approaches to the Diagnosis of Alzheimer Disease
Published in Robert E. Becker, Ezio Giacobini, 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.
The diagnostic significance of CDH17-positive circulating tumor cells in patients with colorectal cancer
Published in Expert Review of Molecular Diagnostics, 2023
Xiao Meng Pei, Heong Ting Wong, Simon Siu Man Ng, Wing Wa Leung, Yee Ni Wong, Hin Fung Tsang, Amanda Kit Ching Chan, Yin Kwan Evelyn Wong, Allen Chi Shing Yu, Aldrin Kay Yuen Yim, William Chi Shing Cho, John Kwok Cheung Chan, Kwong Fai Wong, John M Luk, William Chi Shing Tai, Sze Chuen Cesar Wong
FFPE tissue sections 4um thick of CRC, CRC metastatic to liver and lung, pre-cancerous intestinal metaplasia in stomach, stomach cancer, and pancreatic cancer samples were stained with anti-ARB101 antibody (Arbele Limited, Hong Kong SAR, China) using Leica Bond III automated immunostainer (Leica Microsystems, Wetzlar, Germany). Briefly, sections were washed in Leica Bond Wash Solution followed by antigen retrieval by using BOND Epitope Retrieval Solution 2 for 25 mins at 100°C. After retrieval, the sections were incubated with anti-ARB101 antibody at 1:2000 dilution for 8 minutes followed by anti-human IgG4 at 1:600 dilution for 8 minutes. Colorimetric signal was detected by diaminobenzidine. Percentage of positive cells (ranging from 0 to 100%) and staining intensity score (4: very strong; 3: strong; 2: moderate; 1: weak; 0: negative) were recorded for the calculation of IHC score = percentage of positive cells (0 to 100%) x staining intensity (1 to 4). In brief, the staining intensity was scored as follows: 0 = no expression, 1 = weak expression, 2 = moderate expression, 3 = strong expression, and 4 = very strong expression. The final score was expressed as immunohistochemical staining score (IHC score) obtained by multiplying the percentage of positive cells (or nuclei) with the staining intensity according to our previous method [29]. This scoring method has been widely used to evaluate the results of immunohistochemistry staining [25–28].
Yi Shen An, a Chinese traditional prescription, ameliorates membranous glomerulonephritis induced by cationic bovine serum albumin in rats
Published in Pharmaceutical Biology, 2022
Yun-Li Zhao, Xiang-Hua Zhang, Feng Guo, Ying Wei, Jian-Hua Shang, Xiao-Dong Luo
For immunohistochemical staining, the kidney tissues were fixed using 10% neutral buffered formalin, embedded in paraffin, cut into 4 μm sections, and placed onto glass slides. Next, paraffin was removed with xylene and the sections were dehydrated with a graded series of alcohols, followed by three washes with PBS. Next, the sections were incubated with 3.0% peroxide for 10 min to block the activity of endogenous peroxidase. For antigen retrieval, the kidney sections were immersed in a 10 mmol/L solution of citrate buffer and heated to 90 °C for 20 min. After cooling for 20 min, the sections were incubated in blocking solution (5% BSA) for 20 min at room temperature. After incubation at 37 °C for 2 h, the sections were stained using a biotinylated rabbit anti-rat IgG to rat C3c (Wuhan BOSTER Biological Engineering Co., Ltd., Wuhan, China). After washing with PBS, the slides were incubated with labelled streptavidin-biotin reagent. Immunoreactive products were then visualised using a liquid diaminobenzidine substrate chromogen system. Sections were then counterstained with haematoxylin for 15 s; positive areas showed a granular appearance with linear brown/yellow deposits. The most intense positive area in the cortical zone of each section was measured using an Image-Pro® Plus 6.0 colour pathological image analysis system (Media Cybernetics Co., Ltd., Bethesda USA) at a magnification of ×200 to calculate the integral optical density and mean optical density (Liu et al. 2009; Oktem et al. 2012).
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).