Hamsters were anesthetized and perfused with 2 mL of 10% phosphate-buffered formalin, and the lungs were harvested and fixed. Fixed tissues were routinely embedded in paraffin and sectioned (3 μm). For immunohistochemistry, antigen retrieval of formalin-fixed hamsters tissue sections was performed by autoclaving at 121°C for 10 min in retrieval solution at pH 6.0 (Nichirei, Tokyo, Japan). CoV2 antigens were detected using a standard immunoperoxidase method, and a rabbit anti- CoV2 N antibody was used as the primary antibody (Nagata et al., 2021 (link)). Nuclei were counterstained with hematoxylin for 10 s. Scores were determined based on the percentage of N antigen-positive cells, as determined by immunohistochemistry in each group using the following scoring system: 0, no antigen positive cells; 1, antigen positive cells were occasionally observed in each cut sections (1-3 antigen-positive areas per section were observed in the high magnification); 2, scattered positive cells were observed (4-9 antigen-positive areas per section were observed in the high magnification); 3, many positive cells were diffusely and widely observed (more than 10 antigen-positive areas per section were observed in the low magnification). Mean scores from all lung sections (5-10 lung sections/animal) in each animal were calculated.
Retrieval solution
Manufactured by Nichirei Biosciences
Sourced in Japan
Retrieval solution is a reagent used in immunohistochemistry and in situ hybridization techniques. Its core function is to facilitate the retrieval of target antigens or nucleic acid sequences, which may have been masked or altered during the fixation and processing of biological samples. The solution is designed to unmask the target analytes, making them accessible for subsequent detection and analysis.
Automatically generated - may contain errors
3 protocols using retrieval solution
1
Immunohistochemical Detection of SARS-CoV-2 in Hamsters
2
Immunohistochemical Analysis of Breast Cancer Proteins
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Immunohistochemical analysis of breast cancer tissues was performed with anti-PINK1 (BC100-494, Novus Biologicals, Centennial, CO, USA), anti-Parkin (sc-32282, Santa Cruz Biotechnology), anti-BRCA1 (MS110, Abcam, Cambridge, UK), and anti-Ki67 (MIB-1, M7240, DAKO, CA, USA) antibodies. Paraffin sections were deparaffinized and rehydrated. Antigen retrieval was performed by autoclaving at 105ºC for 20 min with a retrieval solution pH 9.0 (Nichirei Biosciences Inc., Tokyo) for BRCA1, PINK1 and Parkin, or by autoclaving at 121ºC for 10 min with 1 mM Tris/EDTA pH 9.0 for Ki67. For these analyses, tissues showing atypical ductal hyperplasia were excluded. To quantify BRCA1-positive cells, only nuclear signals were used. For the scoring of PINK1 and Parkin expressions in breast cancer tissues, we compared the signal intensity with normal tissues; a signal was counted as negative only when the signal intensity was lower than the intensity in the normal tissues.
3
Immunohistochemical Detection of SARS-CoV-2 in Tissue
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To obtain animal tissues, we anesthetized and perfused mice with 2 ml of 10% phosphate-buffered formalin, and the lungs and head, including the nasal cavity and brain, were harvested and fixed. Fixed tissues were routinely embedded in paraffin, sectioned and stained with haematoxylin and eosin (H&E). For immunohistochemistry, antigen retrieval of formalin-fixed mouse tissue sections was performed by autoclaving at 121 °C for 10 min in retrieval solution at pH 6.0 (Nichirei, Tokyo, Japan). SARS-CoV-2 antigens were detected using a polymer-based detection system (Nichirei-Histofine Simple stain mouse MAX PO; Nichirei Biosciences, Inc., Tokyo, Japan), and an in-house rabbit anti-SARS-CoV-2 N antibody was used as the primary antibody44 (link). Diaminobenzidine (Sigma-Aldrich) was used as the chromogen for HRP visualisation. Nuclei were counterstained with haematoxylin for 10 s. The images were acquired using the imaging software cellSens Standard 2.1 (Olympus).
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