Tumor tissue analogs grown for 10 days under hypoxia received 5 Gy irradiation and then were stained for vimentin on day 5 after treatment. The 4–5 μm TTA sections were placed on the positively charged plus slides (Fisher Scientific, Pittsburgh, PA) and probed with monoclonal mouse anti-vimentin V9 antibody using the automated IHC (Dako, Inc. FLEX System). Briefly, sections were deparaffinized in xylene and hydrated stepwise through ethanol to water. Antigen retrieval was performed using a high pH antigen retrieval buffer (Dako, Inc.) in a decloaking chamber (Biocare Medical, Concord, CA) according to the manufacturer's instructions. Slides were incubated in pre-diluted monoclonal mouse anti-vimentin antibody clone 9 (1:400) for 10 min at room temperature (no. IR630; Dako, Inc.) followed by cell labeling with the EnVision+ kit (Dako, Inc.) according to the manufacturer's instructions. Signal was visualized with 3,3′-diaminobenzidine (DAB) followed by a light hematoxylin counterstain. Immunoprobed sections were then coverslipped with Permount™ and air dried before images were captured with a digital camera (AxioCam, Carl Zeiss Microscopy GmbH, Gottingen, Germany) mounted on an Axioskop 2 Plus microscope (Carl Zeiss Microscopy) at 40× magnification.
High ph antigen retrieval buffer
Manufactured by Agilent Technologies
Sourced in Denmark
The High pH Antigen Retrieval Buffer is a laboratory solution used to prepare samples for immunohistochemical analysis. It is designed to facilitate the exposure of target antigens within fixed tissue samples, which is a critical step in the staining and visualization process.
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2 protocols using high ph antigen retrieval buffer
1
Vimentin Immunohistochemistry of Irradiated Tumor Tissue Analogs
2
Tissue Microarray Construction and Immunohistochemistry
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This study was conducted after receiving approval from the Observational Research Ethics Review Committee of the Osaka University Hospital and Hokkaido University Hospital. Tissue microarray blocks were constructed using a manual tissue microarrayer (JF-4; Sakura Finetek Japan, Tokyo, Japan) with a 2.0-mm diameter needle, from two representative tumor areas (both the invasive front and the bulk of the tumor) and from one representative area of non-neoplastic bile duct as an internal control. The finalized array blocks were sliced into 4-μm-thick sections and mounted onto glass slides. Tissue sections were deparaffinized in xylene and rehydrated through a series of graded ethanol solutions. Heat-induced antigen retrieval was carried out in high-pH antigen retrieval buffer (Dako Cytomation, Glostrup, Denmark). Endogenous peroxidase was quenched with 3% H2O2 for 5 min. These sections were visualized using the HRP-labeled polymer method (EnVision FLEX system, Dako Cytomation). Immunostained sections were counterstained with hematoxylin, dehydrated in ethanol, and cleared in xylene.
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