Ab9377

Manufactured by Abcam

Sourced in United Kingdom, United States

Ab9377 is a laboratory antibody product developed and manufactured by Abcam. It is intended for use in various research and diagnostic applications.

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Lab products found in correlation

34 protocols using ab9377

Rabbit Bladder Tissue Regeneration

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Tissue samples were fixed with 10% neutral formaldehyde, embedded in paraffin, and sectioned in 5 μm diameter specimen, which was followed by HE staining for the observation of material degradation, morphological and histological changes and process of re-epithelialization, and Masson staining for the observation of muscle tissue regeneration.
AE1/AE3 (ab9377, Abcam) antibody was used to observe the re-epithelialization of full-thickness of rabbit bladder. Myosin (ab11083, Abcam) and α-SMA (ab32575, Abcam) were used to observe the regeneration of bladder muscle.

Song Y.T., Li Y.Q., Tian M.X., Hu J.G., Zhang X.R., Liu P.C., Zhang X.Z., Zhang Q.Y., Zhou L., Zhao L.M., Li-Ling J, & Xie H.Q. (2021). Application of antibody-conjugated small intestine submucosa to capture urine-derived stem cells for bladder repair in a rabbit model. Bioactive Materials, 14, 443-455.

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Immunofluorescence Analysis of SARS-CoV-2 Proteins

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Ten-micrometer-thick cryosections were double-stained using a monoclonal mouse anti-SARS-CoV-2 antibody (1:1000 dilution, #MBS569903; MyBioSource, San Diego, CA, USA) and one of the following antibodies: polyclonal rabbit anti-pan cytokeratin antibody (1:100 dilution, #ab9377; Abcam), monoclonal rabbit anti-CD14 antibody (1:100 dilution, #ab18332; Abcam), polyclonal rabbit anti-ACE2 (1:100 dilution, recognizing both short and long forms of ACE2; #PK-AB718-3217, PromoCell, Heidelberg, Germany), monoclonal rabbit anti-ICAM-1 (1:100 dilution, #ab109361, Abcam). Mouse monoclonal and rabbit monoclonal or polyclonal isotype antibodies (#ab18469, #ab172730 and #ab15348; Abcam) functioned as negative controls. In the secondary step, FITC-conjugated polyclonal goat anti-mouse antibodies (1:200 dilution #F2761, ThermoFisher Scientific) were combined with Texas Red-conjugated polyclonal donkey anti-rabbit antibodies (1:100 dilution, #ab6800, Abcam). In the tertiary step, FITC-conjugated polyclonal donkey anti-goat antibodies were added (1:200 dilution, #A16006, ThermoFisher Scientific). DAPI (#D9542, Sigma-Aldrich) was used to counterstain cell nuclei. Slides were mounted with Fluoroshield™ (#F6182, Sigma-Aldrich) and analyzed using a Leica (TCS SPE) confocal microscope and Leica Las X v3.7.2.22383 software.

Van Cleemput J., van Snippenberg W., Lambrechts L., Dendooven A., D’Onofrio V., Couck L., Trypsteen W., Vanrusselt J., Theuns S., Vereecke N., van den Bosch T.P., Lammens M., Driessen A., Achten R., Bracke K.R., Van den Broeck W., Von der Thüsen J., Nauwynck H., Van Dorpe J., Gerlo S., Maes P., Cox J, & Vandekerckhove L. (2021). Organ-specific genome diversity of replication-competent SARS-CoV-2. Nature Communications, 12, 6612.

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Mesangial Cell Characterization by Immunofluorescence

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Human glomerular mesangial IP15 cell lines (HMCs; Renal Disease Center, Royal Free Hospital of England; London, UK) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C with 5% CO₂. The protein expression levels of actin, collagen IV, fibronectin, vimentin, cytokeratin and factor VIII were tested by an indirect immunofluorescence assay using kits for anti-collagen IV antibody (ab6586), anti-β actin antibody (ab8227), anti-fibronectin antibody (ab2413), anti-vimentin antibody (ab137321), anti-wide spectrum cytokeratin antibody (ab9377) and anti-factor VIII antibody (ab203590; all Abcam, Cambridge, UK) according to the previous study (7 (link)).

Deng Y., Lin N., Wu L., Jia Q, & Liu H. (2017). Expression of LOX-1 in human mesangial cells is increased by Ox-LDL and IL-1β treatment. Experimental and Therapeutic Medicine, 14(4), 3632-3636.

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Histological Analysis of Wound Healing

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The collected specimens were fixed in 4% paraformaldehyde solution, dehydrated with a series of graded ethanol, and embedded in paraffin. Sections were stained with Masson trichrome and photographed under an optical microscope. Immunofluorescence staining for CD31 (Abcam, ab76533), vimentin (Cell Signaling, 5741), and cytokeratin (Abcam, ab9377) was performed to assess the extent of newly formed microvessels, fibroblasts, and keratinocytes, respectively. Six random fields per section near wound edges were counted with Image-Pro Plus 6 software.

Liang X., Lin F., Ding Y., Zhang Y., Li M., Zhou X., Meng Q., Ma X., Wei L., Fan H, & Liu Z. (2021). Conditioned medium from induced pluripotent stem cell-derived mesenchymal stem cells accelerates cutaneous wound healing through enhanced angiogenesis. Stem Cell Research & Therapy, 12, 295.

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Protein Expression Analysis in Tumor Cells

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Proteins were extracted from cultured cells, and tumor tissues of tumor-bearing mice using, respectively, 0.1 ml and 1.0 ml radioimmunoprecipitation assay reagent containing 1% phenylmethanesulfonyl fluoride to prevent protein degradation. Protein extracts were subjected to SDS-PAGE and analyzed using the following primary antibodies: TGF-β1, Smad2/3 (sc-398844, Santa Cruz Biotechnologies), P-smad2/3, E-cadherin (ab1416, Abcam), N-cadherin (ab6528, Abcam), cytokeratin (CK) (ab9377, Abcam), β-catenin (ab32572, Abcam), α-SMA (ab124964, Abcam), MMP-7 (ab5706, Abcam), and β-actin (TA-09; ZSGB-BIO, Beijing China). After that, the membranes were incubated with secondary antibodies at 4°C overnight, washed in PBST thrice for 10 min each time, and incubated with the corresponding secondary antibody. The dilution ratio was determined as recommended. Protein levels were measured using the Image-Pro Plus system version 6.0. Band intensity was quantified using the ImageJ software version 1.34. All experiments were performed in triplicate.

Shi Q., Wu H., Li Y., Shen L., Tian X., Lin T, & Wei G. (2020). Inhibition of Wilms' Tumor Proliferation and Invasion by Blocking TGF-β Receptor I in the TGF-β/Smad Signaling Pathway. BioMed Research International, 2020, 8039840.

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Multiplex Immunohistochemistry of Melanoma

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Formalin-fixed, paraffin-embedded primary melanoma tumor biopsies were processed in the BondRX for OPAL staining (Perkin-Elmer, Waltham, MA) using the ER2 instrument protocol for 20 min at 100°C with Epitope Retrieval Solution (pH 6 EDTA-based retrieval solution) followed by probing with rabbit anti-EOMES-Ac, anti-EOMES-Me2, or pan-cytokeratin (Ab9377; Abcam, Cambridge, UK) and mouse host CD8 (provided by the Opal kit) antibodies and visualized with Opal kit 520, 570, 650, and 690. Coverslips were mounted on glass microscope slides with ProLong Clear Antifade reagent (Life Technologies, Carlsbad, CA). The 7-color Automation kit (NEL801001KT), 7-color Immune Discovery kit (OP7DS2001KT), 4-color Automation kit (NEL820001KT), and Opal 4 Lymphocyte kit (OP4LY2001KT) were used.

Tu W.J., McCuaig R.D., Tan A.H., Hardy K., Seddiki N., Ali S., Dahlstrom J.E., Bean E.G., Dunn J., Forwood J., Tsimbalyuk S., Smith K., Yip D., Malik L., Prasanna T., Milburn P, & Rao S. (2020). Targeting Nuclear LSD1 to Reprogram Cancer Cells and Reinvigorate Exhausted T Cells via a Novel LSD1-EOMES Switch. Frontiers in Immunology, 11, 1228.

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Immunostaining of Patient-Derived PDAC Cells

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(Figure S1A) Patient-derived PDAC-3 and CAF-1 cells were co-cultured in chambered slides (Millicell® EZ slide Cat.No: PEZGS0416) and immunostained using a primary-secondary approach. Cell were washed three times with PBS and fixed with 4% paraformaldehyde for 5 min, washed 3 times with PBS for 5 min each, blocked with 5% normal goat serum in PBS per 30 min, permeabilized with 0.3% TWEEN 20 for 5 min, and stained primary antibodies were rabbit anti-wide spectrum cytokeratin (1:50, Abcam ab9377) and mouse anti-actin a-smooth muscle actin (1:500, Sigma A2547). Secondary immunofluorescent-tagged antibodies were used for signal amplification. Goat antirabbit IgG Alexa Fluor 488 and goat anti-mouse IgG 594 were used for secondary amplification. Nuclei were then counterstained with nuclear 4,6-diamidino-2-phenylindole (DAPI) and the slides were rinsed with PBS, cover slipped and stored at 4°C. Fluorescence images were acquired using a stan dard up-right fluorescent microscope (Nikon 90-I eclipse). GFP and mCherry protein was detected in PDAC-3 and CAF-1 cells, respectively, in co-culture in chambered slides.

Ligorio M., Sil S., Malagon-Lopez J., Nieman L.T., Misale S., Di Pilato M., Ebright R.Y., Karabacak M.N., Kulkarni A.S., Liu A., Jordan N.V., Franses J.W., Philipp J., Kreuzer J., Desai N., Arora K.S., Rajurkar M., Horwitz E., Neyaz A., Tai E., Magnus N.K., Vo K.D., Yashaswini C.N., Marangoni F., Boukhali M., Fatherree J.P., Damon L.J., Xega K., Desai R., Choz M., Bersani F., Langenbucher A., Thapar V., Morris R., Wellner U.F., Schilling O., Lawrence M.S., Liss A.S., Rivera M.N., Deshpande V., Benes C.H., Maheswaran S., Haber D.A., Fernandez-Del Castillo C., Ferrone C.R., Haas W., Aryee M.J, & Ting D.T. (2019). Stromal Microenvironment Shapes the Intratumoral Architecture of Pancreatic Cancer. Cell, 178(1), 160-175.e27.

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Comprehensive Immunohistochemistry Panel

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Primary antibodies: Rabbit anti-cleaved Caspase 3 (9661S, Cell Signaling Technology, Danvers, USA), rabbit anti-Ki-67 (ab16667, Abcam, Cambridge, UK), rat anti-CD31 (DM3614P, Dianova, Hamburg, Germany), goat anti-CD32b (AF1460, R&D Systems, Minneapolis, USA), goat anti-Lyve1 (AF2125, R&D Systems, Minneapolis, USA), rat anti-Endomucin (14–5851–82, Thermo Fisher Scientific, Waltham, USA), rabbit anti-TRP-2 (ab74073, Abcam, Cambridge, UK), rabbit anti-wide spectrum cytokeratin (ab9377, Abcam, Cambridge, UK), goat anti-alpha smooth muscle actin (ab21027, abcam, Cambridge, UK), rat anti-CD45 (550539, BD Biosciences, Franklin Lakes, USA), rat anti-CD68 (137002, BioLegend, San Diego, USA), rabbit anti-Melan A (NBP1-30151, Novus, Minneapolis, USA). For Western blotting: rabbit anti-Akt (9272S, Cell Signaling Technology, Danvers, USA), rabbit anti-phospho-Akt (9271S, Cell Signaling Technology, Danvers, USA). Secondary antibodies: Alexa-Fluor 488, Alexa-Fluor 647 and Cy3-conjugated secondary antibodies were purchased from Dianova (Hamburg, Germany). For Western Blotting a rabbit anti-IgG HRP conjugated antibody was used. (Merck, Darmstadt, Germany).

Dietsch B., Weller C., Sticht C., de la Torre C., Kramer M., Goerdt S., Géraud C, & Wohlfeil S.A. (2023). Hepatic passaging of NRAS-mutant melanoma influences adhesive properties and metastatic pattern. BMC Cancer, 23, 436.

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Immunohistochemical Characterization of Lung Metastases

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Paraffin embedded tissue blocks were prepared, sectioned, and stained using standard immunohistochemical techniques by the Sanford Molecular Pathology Core. Briefly, paraffin embedded tissues were sectioned to 5 μm and stained on a BenchMark^® XT automated slide stainer (Ventana Medical Systems, Inc). Ventana iView DAB detection kit and counterstaining with hematoxylin were used for visualization. Staining for pan-cytokeratin (ab9377, Abcam), E-cadherin (#3195, Cell Signaling), P16 (MA5-17093, Thermo and #10883-1-AP, ProteinTech), BRCA2 (HPA026815, Sigma), and EGFR (ab2430, Abcam) were used to visualize tumors of epithelial origin; exclusion of primary antibody served as the negative control. Lung metastases from three independent sections were manually counted on an (Olympus DP71) microscope.

Vermeer D.W., Coppock J.D., Zeng E., Lee K.M., Spanos W.C., Onken M.D., Uppaluri R., Lee J.H, & Vermeer P.D. (2016). Metastatic model of HPV+ oropharyngeal squamous cell carcinoma demonstrates heterogeneity in tumor metastasis. Oncotarget, 7(17), 24194-24207.

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Immunohistochemical detection of cytokeratin

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Tissue sections were deparaffinized and rehydrated as mentioned earlier. Then sections were placed in 3% H₂O₂ for 15 min to remove the endogenous enzymes and washed three times in PBS for 5 min at room temperature. Next, 50 μl trypsin was added to each sample, and the antigen was repaired in 37°C incubators for 30 min. After being washed in PBS 3 times, the sections were blocked with 5% BSA for 30 min at 37°C. Subsequently, the slides were exposed to anti-wide spectrum cytokeratin (1:200, ab9377, Abcam) primary antibody at 4°C overnight. After being rinsed with PBS for three times, the sections were incubated with secondary antibodies (1:1000, ab150077, Abcam) for 1 h at 37°C and further developed with 3,3′-diaminobenzidine tetrahydrochloride (DAB) solution. Finally, sections were counterstained with hematoxylin for 2 min, followed by 3 min in PBS to reduce the background staining. Positive staining was indicated by brown color, which can be observed under a Nikon confocal laser microscope (A1 PLUS, Nikon Tokyo, Japan).

Zhu H., Xu J., Zhao M., Luo H., Lin M., Luo Y., Li Y., He H, & Wu J. (2022). Adhesive, injectable, and ROS-responsive hybrid polyvinyl alcohol (PVA) hydrogel co-delivers metformin and fibroblast growth factor 21 (FGF21) for enhanced diabetic wound repair. Frontiers in Bioengineering and Biotechnology, 10, 968078.

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