Ab236466

Manufactured by Abcam

Sourced in United States, United Kingdom

Ab236466 is a lab equipment product. It is a device used for scientific research and analysis purposes.

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8 protocols using ab236466

Immunohistochemistry Analysis of Mouse Leg Tissue

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For IHC staining, the decalcified mouse leg tissue samples were prepared as above. Cryostat sectioning was performed on the frozen tissue samples to obtain about 5 µm-thick tissue sections, which were left at room temperature overnight. The tissue sections were fixed in 4% paraformaldehyde for 15 min and then the sections were incubated overnight at 4 °C with rabbit anti-α-SMA antibody (1:500; Cat. #ab124964, Abcam; Waltham, MA, USA), rabbit anti-TGF-β1 antibody (1:500; Cat. #ab215715, Abcam; Waltham, MA, USA), rabbit anti-Scx antibody (1:500; Cat. #ab58655, Abcam, Waltham, MA, USA), or rabbit anti-HMGB1 antibody (1:330; Cat. #ab18256, Abcam; Waltham, MA, USA). The positively stained results were tested using a rabbit-specific HRP/DAB IHC detection kit (Cat. #ab236466; Abcam, Waltham, MA, USA).
For AMPK activation testing, the fixed tissue sections were treated with 0.1% Triton X-100 at 37 °C for 30 min, then washed with PBS 3 times. The treated tissue sections were incubated with rabbit anti-AMPK antibody (1:500, Cat. #MA5-15815, ThermoFisher Scientific; Waltham, MA, USA) or with rabbit anti-phospho-AMPK antibody (1:500, Cat. #ab133448, Abcam; Waltham, MA, USA) at 4 °C overnight. The staining results were further tested using a rabbit-specific HRP/DAB IHC detection kit (Cat. #ab236466; Abcam, Waltham, MA, USA).

Zhang J., Brown R., Hogan M.V, & Wang J.H. (2023). Mitigating Scar Tissue Formation in Tendon Injuries: Targeting HMGB1, AMPK Activation, and Myofibroblast Migration All at Once. Pharmaceuticals, 16(12), 1739.

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Histological Analysis of Liver and Adipose Tissues

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Liver and adipose tissues were fixed in 4% formalin and embedded in optimal cutting temperature compound or paraffin. Oil red O staining or H&E staining was performed as descried previously.⁴⁵ (link) The terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling assay was performed using an In-Situ Cell Death Detection Fluorescein kit (11684795910; Roche, St. Louis MO). Sirius red staining was performed using Picro Sirius Red Solution (ab246832; Abcam, Cambridge, MA). For immunostaining, sections were incubated with a rabbit anti–cleaved caspase-3 antibody (Asp175, 5A1E; Cell Signaling Technology, Danvers, MA) or UCP1 (10983; Abcam) overnight at 4°C and then incubated with a horseradish-peroxidase–conjugated secondary antibody using a mouse- and rabbit-specific horseradish-peroxidase/3,3′-diaminobenzidine tetra hydrochloride immunohistochemistry detection kit micropolymer (ab236466; Abcam). Hematoxylin was used for the counterstaining of nuclei.

Fan M., Wang Y., Jin L., Fang Z., Peng J., Tu J., Liu Y., Zhang E., Xu S., Liu X., Huo Y., Sun Z., Chao X., Ding W.X., Yan Q, & Huang W. (2021). Bile Acid–Mediated Activation of Brown Fat Protects From Alcohol-Induced Steatosis and Liver Injury in Mice. Cellular and Molecular Gastroenterology and Hepatology, 13(3), 809-826.

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Evaluating Tumor Stromal CD163 Expression

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Formalin-fixed paraffin-embedded (FFPE) tissue sections were obtained from breast cancer patients. The antigen unmasking was performed using the PT Link module (Dako, Denmark) in T/E buffer (pH 9.0). Immunohistochemical staining was performed using monoclonal rabbit anti-CD163 (1:500, ab182422, Abcam) and visualized using the Polymer-HRP detection system (ab236466, Abcam, USA). The staining results were acquired by a Carl Zeiss Axio Lab.A1 light microscope (Jenamed, Carl Zeiss, Germany) and assessed as the percentage of area occupied by positive stromal cells over the total intratumoral stromal area (according to Salgado et al.) (31 (link)). Cells outside of the tumor border and around DCIS and normal lobules, as well as in tumor zones with crush artifacts, necrosis, and regressive hyalinization, were excluded.

Patysheva M., Larionova I., Stakheyeva M., Grigoryeva E., Iamshchikov P., Tarabanovskaya N., Weiss C., Kardashova J., Frolova A., Rakina M., Prostakishina E., Zhuikova L., Cherdyntseva N, & Kzhyshkowska J. (2022). Effect of Early-Stage Human Breast Carcinoma on Monocyte Programming. Frontiers in Oncology, 11, 800235.

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Immunohistochemical Analysis of Ovarian Cancer

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Cytospins were prepared with cellularized tumor cells for immunohistochemical studies, as described previously.28 Then, cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.3% Triton X-100 in PBS for 30 min, and blocked with 5% normal goat serum for 1 h at room temperature. The cytospins were then incubated at 4°C overnight with primary rabbit/mouse monoclonal antibodies generated against the following proteins: WT1, a protein that is expressed at high levels in patients with epithelial ovarian cancer (1:600, ab89901, Abcam, Cambridge, UK); MUC16, a transmembrane glycoprotein expressed at high levels in over 80% of patients with ovarian cancer; CA125 (1:2000, ab110640, Abcam); and Ki67, as an indicator of tumor proliferation (1:400, ab245113, Abcam). Subsequently, a goat anti-rabbit secondary antibody (ab236466, Abcam) was applied for 10 min at room temperature; a mouse-specific reagent was incubated for 10 min prior to this step if a mouse primary antibody had been applied. Immunoreactive signals were visualized by incubation in diaminobenzidine (DAB) for 5 min. Finally, we captured images with a microscope (Leica DMI3000 B, Germany) and edited them with Photoshop (Adobe, USA).

Huang Y., Xu J., Li K., Wang J., Dai Y, & Kang Y. (2021). A Novel, Personalized Drug-Screening System for Platinum-Resistant Ovarian Cancer Patients: A Preliminary Clinical Report. Cancer Management and Research, 13, 2849-2867.

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Multimodal Molecular Imaging Protocol

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The 4-0 suture was purchased from UNIK SURGICAL SUTURES MFG. CO., LTD. (New Taipei City, Taiwan). The Picro Sirius Red kit was purchased from Abcam (ab150681, Cambridge, UK). The FEPPA precursor (no. TEPP-90-0005) was purchased from Huayi Isotope Company (Shanghai, China). The [¹⁸F]FDG was kindly provided by Taipei Veterans General Hospital (Taipei, Taiwan). All other chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). GENEzol TriRNA Pure kit was purchased from Geneaid Biotech Ltd. (New Taipei City, Taiwan). Primary anti-GLUT1 (ab115730) and anti-TSPO (ab109497) antibodies were purchased from Abcam (Cambridge, UK). The secondary antibody was purchased from Thermo Fisher Scientific (New York, USA). The mounting medium was purchased from Abcam (ab236466, Cambridge, UK).

Wu C.Y., Hsieh H.H., Chu P.A., Hong W.H., Chang T.Y., Hsu C.F., Lin S.T., Su P.H, & Peng S.L. (2021). Comparison of 18F-FDG, 18F-Fluoroacetate, and 18F-FEPPA for Imaging Liver Fibrosis in a Bile Duct-Ligated Rat Model. Molecular Imaging, 2021, 7545284.

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Immunohistochemical Analysis of p62 in Tumors

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IHC was performed as mentioned earlier^{59 (link)}. Briefly, 5-µm thick sections of tumors were deparaffinised, hydrated, and treated with peroxide (Abcam, UK ab236466), which was followed by heat induced epitope retrieval in sodium isocitrate buffer (pH 8 for p62). For p62, antigen retrieval was carried at 120 °C for 6 min in a pressure cooker. The sections were then incubated with the protein blocking reagent (Abcam, UK) and treated with anti-p62 (Abcam, UK; 1:100; 4 °C for overnight). The sections were further treated with the secondary antibodies and developed using HRP-conjugated DAB substrate (Abcam, UK). Grading was done based on the intensity and the extent of positivity as scored by an experienced pathologist.

Bishnu A., Phadte P., Dhadve A., Sakpal A., Rekhi B, & Ray P. (2021). Molecular imaging of the kinetics of hyperactivated ERK1/2-mediated autophagy during acquirement of chemoresistance. Cell Death & Disease, 12(2), 161.

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Immunohistochemical Detection of p-STAT3

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The immunohistochemistry staining followed the manufacturer's protocols, and the heat-induced epitope retrieval method with citrate buffer was used for antigen retrieval. Primary antibodies, specifically anti-STAT3 (Y705) antibody (rabbit polyclonal, ab214465 (dilution 1:500), Abcam), were added and incubated at 4°C overnight. The primary antibodies were detected using a mouse and rabbit specific HRP/DAB IHC detection kit (ab236466, Abcam) following the manufacturer's instructions.

Tang Y., Tan S.A., Iqbal A., Li J, & Glover S.C. (2019). STAT3 Genotypic Variant rs744166 and Increased Tyrosine Phosphorylation of STAT3 in IL-23 Responsive Innate Lymphoid Cells during Pathogenesis of Crohn's Disease. Journal of Immunology Research, 2019, 9406146.

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Histopathological Analysis of Mouse Organs

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The organs of the mice (lung and spleen) were collected after euthanasia and prepared for histopathological examination. Tissues were fixed in buffered 10% neutral formalin, dehydrated, embedded in paraffin wax, and sectioned on a microtome at a thickness of 4 μm. Tissue sections from the organs of all mice were stained with hematoxylin and eosin (H&E). A Warthin–Starry (WS) silver staining kit (010270, Diapath S.p.A., Martinengo, Italy) was used for the proof of bacteria. Masson’s trichrome staining was performed to stain collagen connective tissue. In addition, immunohistochemistry was conducted on the samples to confirm the presence of macrophages (CD11b+). For this, rabbit monoclonal anti-CD11b antibody (1/4000 dilution, ab133357, Abcam, Cambridge, United Kingdom) and a Specific HRP/DAB IHC detection kit micropolymer (ab236466, Abcam) were used according to the manufacturer’s conditions.

Trousil J., Frgelecová L., Kubíčková P., Řeháková K., Drašar V., Matějková J., Štěpánek P, & Pavliš O. (2022). Acute Pneumonia Caused by Clinically Isolated Legionella pneumophila Sg 1, ST 62: Host Responses and Pathologies in Mice. Microorganisms, 10(1), 179.

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