Ab78196

Manufactured by Abcam

Sourced in United States, China, United Kingdom

Ab78196 is a laboratory equipment product. It serves a core function in research and scientific applications, but a detailed description cannot be provided while maintaining an unbiased and factual approach.

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Lab products found in correlation

5 protocols using ab78196

Histological Analysis of Neobladder Tissues

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After physiology test, blood and urine sample collection, pigs were euthanized, and their neobladders were harvested for histological analysis. The harvested neobladder tissues were fixed in 10% formalin for 1 week, then embedded with paraffin and sliced into 5 μm sections. Haematoxylin and eosin (H&E) stains were performed according to standard procedures. Immunohistochemical (IHC) analysis was performed using the mouse monoclonal antibody against cytokeratin 7 (CK7) (1:100, ab9021, Abcam), Uroplakin III (UPK3) (1:20, ab78196, Abcam) and alpha smooth muscle actin (α-SMA) (1:200, A5228, Sigma-Aldrich). The staining was performed according to the manufacturer of the mouse and rabbit specific HRP/DAB detection IHC kit (ab64264, Abcam). Images were obtained using the Pannoramic Scanner system (3D HISTECH, Ltd., Budapest, Hungary).

Chen B., Chen X., Wang W., Shen J., Song Z., Ji H., Zhang F., Wu J., Na J, & Li S. (2021). Tissue-engineered autologous peritoneal grafts for bladder reconstruction in a porcine model. Journal of Tissue Engineering, 12, 2041731420986796.

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Western Blot Analysis of Cellular Proteins

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Whole cell extracts were prepared with a cell lysis reagent (Sigma-Aldrich; Merck KGaA) according to the manual, and the protein was quantified by a BCA assay (Pierce; Thermo Fisher Scientific, Inc.). Protein extracts were separated using SDS/polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were hybridized with anti-β-catenin antibody (dilution 1:500, ab32572; Abcam), TGFR1 (dilution 1:2,000, ab31013; Abcam), CK7 (dilution 1:500, ab9021; Abcam), CK20 (dilution 1:1,000, ab76126; Abcam), UPIII (dilution 1:800, ab78196; Abcam) and anti-β-actin antibody (dilution 1:800, sc-47778; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), at 4°C overnight. After extensive washing three times for 10 min each in TBS-T (Sigma-Aldrich; Merck KGaA), secondary biotin-conjugated antibodies were added for 1 h at 22°C. Blots were again washed three times for 10 min each in TBS-T, and immunoreactive bands were developed by an avidin/biotin/peroxidase complex (Vectastain ABC Elite kit; Vector Laboratories lnc., Burlingame, CA, USA) and substrate (Vector NovaRED, Vectastain; Vector Laboratories lnc.).

Fan G., Xu Z., Hu X., Li M., Zhou J., Zeng Y, & Xie Y. (2017). miR-33a hinders the differentiation of adipose mesenchymal stem cells towards urothelial cells in an inductive condition by targeting β-catenin and TGFR. Molecular Medicine Reports, 17(2), 2341-2348.

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Immunohistochemical Characterization of Tissue

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Tissue samples were rinsed with 0.9% NaCl, then fixed with 10% formalin, gradient alcohols was dehydrated and paraffin-embedded, sectioned at 3–5 μm for H&E staining. Immunofluorescence staining was also performed with various primary antibodies including AE1/AE3 (1:500 diluted, ab9377, Abcam), Uroplakin III (1:100 diluted, ab78196, Abcam), α-SMA (1:200 diluted, ab32575, Abcam), Myosin (1:500 diluted, ab11083, Abcam), SYP38 (1:100 diluted, ab8049, Abcam), and CD31 (1:100 diluted, ab199012, Abcam), followed by incubation of the secondary antibodies as well as DAPI. Image Pro Plus was applied to analyze the images, including the percentage of positive staining area, and the number of vessels.

Zhang X.Z., Jiang Y.L., Hu J.G., Zhao L.M., Chen Q.Z., Liang Y., Zhang Y., Lei X.X., Wang R., Lei Y., Zhang Q.Y., Li-Ling J, & Xie H.Q. (2020). Procyanidins-crosslinked small intestine submucosa: A bladder patch promotes smooth muscle regeneration and bladder function restoration in a rabbit model. Bioactive Materials, 6(6), 1827-1838.

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Immunostaining of Epithelial Markers

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Cells were fixed with 4% paraformaldehyde for 10 min at room temperature and with methanol at −20°C for 20 min. Normal goat serum (10%; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) was added to cells for 30 min to block nonspecific binding sites. The fixed cells were immunostained with primary antibodies targeting CK7 (dilution 1:500, ab9021; Abcam), CK20 (dilution 1:300, ab76126; Abcam) and UPIII (dilution 1:500, ab78196; Abcam) overnight at 4°C and the Alexa Fluor 488-conjugated secondary antibody (1:500 dilution, SP-9000; ZSGB-BIO, Beijing, China) for 1 h at 37°C. The coverslips were stained with DAPI (1:1,000, SC-3598; Santa Cruz Biotechnology, Inc.) for 2 min and mounted on slides using anti-fade mounting medium. Images were acquired with a high-resolution CoolSNAP™ CCD camera (Photometrics Inc., Tucson, AZ, USA) under the control of a computer using Leica FW4000 software version 1.2 (Leica Microsystems, Ltd., Milton Keynes, UK).

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Immunohistochemical Analysis of Bladder Tissue

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Paraffin sections were cut (5-mm thick), dewaxed in xylene, and treated with an ethanol gradient. The sections were stained with hematoxylin and eosin or with antibodies against keratin 5 (Krt5) (Ab52635, 1:100; Abcam, Cambridge, UK), keratin 14 (Krt14) (Ab7800, 1:200; Abcam), forkhead box A1 (Foxa1) (Ab170933, 1:200; Abcam), and epidermal growth factor receptor (Ab52894, 1:200; Abcam).
For immunofluorescence, antigen retrieval was performed with citrate buffer (pH 6) before primary antibody incubation at 4 C overnight. Sections were stained with primary antibodies against Upk (Ab78196, 1:200; Abcam), Krt5 (Ab52635, 1:100; Abcam), and RFP (Ab62341, 1:50; Abcam), and secondary antibodies Alexa Fluore488 and Alexa Fluore594 (10 mg/mL; Thermo Fisher Scientific, Paisley, UK) for 1 hour at room temperature. Slides were mounted with Mowiol 488 (Sigma-Aldrich, Dorset, UK) containing DAPI (Vectashield Mounting Medium with DAPI, H-1200; Vector Laboratories, Burlingame, CA). Images were taken on a Leica confocal laser microscope (TCS SP8; Leica Microsystems, Wetzler, Germany).
Histopathologic evaluation of the bladder tissues was performed in a blind fashion by one pathologist at Kyoto Histopathology Study Group, Inc., and by two trained urologists (N.M. and T.K.), based on the 2004 World Health Organization Classification of Bladder Tumors.

Masuda N., Murakami K., Kita Y., Hamada A., Kamada M., Teramoto Y., Sakatani T., Matsumoto K., Sano T., Saito R., Okuno Y., Ogawa O, & Kobayashi T. (2020). Trp53 Mutation in Keratin 5 (Krt5)-Expressing Basal Cells Facilitates the Development of Basal Squamous-Like Invasive Bladder Cancer in the Chemical Carcinogenesis of Mouse Bladder. The American journal of pathology, 190(8).

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