Protein were extracted from mice tissue in radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China), proteins were separated by SDS-PAGE and were visualized using ECL system (Bio-Rad, Hercules, CA, United States). Primary antibodies used in this study were as follows: Occludin Polyclonal Antibody (71–1,500) and ZO-1 Polyclonal Antibody (71–1,500) were purchased from Invitrogen (Carlsbad, CA, United States). IRS-1 (D23G12) Rabbit mAb (3407), Phospho-IRS-1 (Ser307) Antibody (2381), mTOR (7C10) Rabbit mAb (2983), Phospho-mTOR (Ser2448; D9C2) XP Rabbit mAb (5536) were purchased from Cell Signaling Technology (Danvers, MA, United States), Monoclonal Anti-β-Actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, United States).
Zo 1 polyclonal antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ZO-1 Polyclonal Antibody is a laboratory research tool used to detect the tight junction protein ZO-1 in various cell and tissue samples. It is designed for immunohistochemistry, immunocytochemistry, and Western blot applications.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (3 mentions)
Anti β actin monoclonal antibody, by Merck Group (1 mentions)
Ecl system, by Bio-Rad (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Electronic balance, by Mettler Toledo (1 mentions)
Inverted fluorescence microscope, by Nikon (1 mentions)
Triton x 100, by Solarbio (1 mentions)
Anti actin, by Merck Group (1 mentions)
Celltracker green, by Thermo Fisher Scientific (1 mentions)
Celltracker red, by Thermo Fisher Scientific (1 mentions)
Anti β actin antibody, by Proteintech (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Biotinylated goat anti rabbit, by Agilent Technologies (1 mentions)
Streptavidin fluorescein isothiocyanate fitc, by BioLegend (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Anti asc al 177, by Adipogen (1 mentions)
9 protocols using zo 1 polyclonal antibody
1
Western Blot Analysis of Tight Junction and Insulin Signaling
2
Immunofluorescent Staining of Epithelial Cell Tight Junctions
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Upon completion of the epithelium generation in the Mesh device, the HCT-116 cell monolayer was fixed in 4% paraformaldehyde at room temperature (RT) for 30 min. Subsequently, cells were washed three times with PBS and permeabilized with 0.1% Triton X-100 in PBS for 15 min. The culture was subjected to three washes in abundant 0.05% Tween20 in PBS and further incubated in a blocking solution (3% BSA (A9418-506, Sigma-Aldrich) in PBS) for 3 h at RT. Following this, cells were washed three times with 0.5% BSA in PBS and labeled with an anti-ZO1 primary antibody (ZO-1 Polyclonal Antibody, Invitrogen) diluted 1:200 in 0.5% BSA in PBS. The incubation with the primary antibody was conducted overnight at RT. After the primary antibody incubation, cells were washed five times with 0.5% BSA (in PBS) before being incubated with a conjugated secondary antibody (Alexa Fluor 555, Invitrogen, A21428) diluted 1:500 in PBS for 2 h at RT. Finally, after three washes with 0.5% BSA) and then three times with PBS, nuclei staining with Hoechst (Hoechst 33342, Invitrogen) was performed by adding the solution to the cells at a final concentration of 1 ug mL−1, followed by incubation at RT for an additional 30 min.
3
Quantifying Cellular Tight Junctions
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Bovine serum albumin (BSA, Sigma), ZO-1 Polyclonal Antibody (Invitrogen, USA), Triton X-100 (Solarbio, China), Alexa Fluor™ 594 (Invitrogen, USA), 34 incubator (Shanghai Yiheng Technology Co., Ltd., China), conversion fluorescence microscope (Olympus, Japan), inverted fluorescence microscope (Nikon, Japan), electronic balance (Mettler Toledo Inc, China), ultra-clean
4
Immunofluorescence and Western Blotting Protocols
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Most reagents including Cell-Tracker Red (CMPTX) cat #C34552 and Cell-Tracker Green (CMFDA) cat #C7025 were obtained from Thermo Fisher. ZO-1 Polyclonal Antibody (Invitrogen, Waltham, MA, USA, cat #40−2200) and M-Sec/TNFAIP2 (Abcam, Cambridge, UK, ab91235) antibodies were used for immunofluorescence and anti-actin (Sigma, Burlington, MA, USA) or anti-TNFAIP2 (Invitrogen, Waltham, MA, USA, PA5-13542) were used in western blotting.
5
Western Blot Analysis of Tight Junction Proteins
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The total proteins of tissues were obtained using RIPA Lysis Buffer (Solarbio Life Science, Beijing, China) and quantified. Thereafter, protein was denatured and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was treated overnight at 4°C with primary antibody before being incubated for 60 min at 22°C with the secondary antibody. The bands were detected using an improved chemiluminescent substrate (Millipore, USA). Antibodies: anti-β-actin antibody (1:10,000; Proteintech, Hubei, China), ZO-1 Polyclonal Antibody (1:500; Thermo Fisher Scientific, USA, 61-7300), Occludin Polyclonal Antibody (1:200; Thermo Fisher Scientific, USA, 71-1500), Claudin-1 Polyclonal Antibody (1:250; Thermo Fisher Scientific, USA, 51-9000), goat anti-mouse IgG (1:1,000; Zhongshan GoldBridge, Beijing, China), and goat anti-rabbit IgG (1:1,000; Zhongshan Gold Bridge, Beijing, China).
6
Immunofluorescence Staining of Tight Junction Proteins
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The slides were collected after incubation with hMOs or hMO-treated bacteria. The cells were washed twice with 0.01% CaCl2, and fixed with ice-cold acetone/methanol (1:1, v/v) for 5 minutes at −20°C. Afterwards, cells were washed 3 times with PBS and blocked with 10% goat serum in 1% BSA for 1 h at room temperature. After overnight incubation with the primary antibody for ZO-1 (ZO-1 Polyclonal Antibody, 1:200, Thermo Fisher Scientific) and claudin-3 (claudin-3 Polyclonal Antibody, 1:50, Thermo Fisher Scientific) at 4°C, the cells were washed 3 times with PBS, and incubated with secondary antibody biotinylated goat anti-rabbit (1:500, Dako) for 1 h at room temperature. The cells were then washed 3 times with PBS and labeled with Streptavidin fluorescein isothiocyanate (FITC) (1:500, BioLegend) in the dark for 1 h. The cell nuclei were stained with DAPI (1:5000, Sigma) followed by 3 washes with PBS.
7
Immunocytochemical Analysis of ZO-1 in HCEn
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To evaluate the expression of ZO-1 marker by HCEn cultured on GELGYM, we used standard ICC assay. Briefly, HCEn (10000 cells per well) were seeded on GELGYM culture discs with varying CT (3, 5, and 10 min). At confluency, the discs were removed from media, carefully rinsed with PBS, and fixed in paraformaldehyde (4%). For permeabilization and blocking, discs were incubated in PBS containing Triton X-100 (0.25%) for 10 min and FBS (5%) in PBS containing Tween-20 (0.05%) for 1 h, respectively. The discs were then incubated with the rabbit polyclonal antibody against ZO-1 (ZO-1 Polyclonal Antibody, dilution: 1:100, ThermoFisher Scientific) as a primary antibody overnight at 4 °C in humidifying condition. Afterward, the specimens were incubated with Cy5-conjugated anti-Rabbit antibody (dilution: 1:200, abcam) as a secondary antibody for 1 h at room temperature. Samples were stained with DAPI (Vector Laboratories) for nuclear staining. Images were taken by a fluorescent microscope (Axio Observer Z1, Zeiss).
8
Immunofluorescence Staining of bEnd5 Cells
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For histology we fixated bEnd5 cultures with a glyoxal solution containing 40% glyoxal, acetic acid, water, and 100% ethanol. Cell cultures were dyed with DAPI, PI, and antibodies against NLRP3 (anti-NLRP3/NALP3, mAb (Cryo-2), #AG-20B-0014, 1:100, Adipogen Life Sciences, San Diego, CA, USA), Zonula occludens 1 (ZO-1 Polyclonal Antibody, #61-7300, 1:1000, Thermo Fisher Scientific), and ASC (Anti-ASC (AL177), #AG-25B-0006, 1:100, Adipogen). We used secondary antibodies in a dilution of 1:100. For recording, we used a Leica microscope (Leica DMi8, DMC 2900/DFC 3000G camera control, LAS X software (Leica, Wetzlar, Germany)). To measure cell death bEnd5 cells cultivated on 96-well plates were visualized with transmitted light microscopy and apoptotic cells with fluorescence measurements after PI (1:200) staining with the above-mentioned microscope. The red fluorescent cells were counted. For measurement of NLRP3, ASC, and ZO-1 intensity, images of the cell cultures were recorded with the same microscope. Subsequently, after converting the images into 16-bit black and white files, the intensities of the respective stainings were determined with ImageJ Analysis Software 1.52a (National Institutes of Health, Bethesda, MD, USA).
9
Urothelial Integrity and Inflammation Assessment
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The grade of epithelial integrity and epithelial damage was assessed by the uroplakin-III (UP-III) polyclonal antibody, the E-cadherin monoclonal antibody (Cell Marque, Rocklin, CA, USA), and the zonula occludens-1 (ZO-1) polyclonal antibody (Thermo Scientific, Waltham, MA, USA), while the severity of inflammation was evaluated by the interleukin-8 (IL-8) antibody (Novus Biologicals, Centennial, CO, USA) with an immunohistochemical evaluation. Each specimen was assessed separately at ×40 magnification. The assessment was carried out using an Olympus BX51 light microscope and an Olympus DP72 digital camera system. Normal expression of UP-III, E-cadherin, and ZO-1 was defined as even distribution throughout the urothelium, mainly in the surface of the epithelial cells. Abnormal expression was defined as weak expression and distribution throughout the urothelium. Specimens with no IL-8 expression, IL-8 expression with mild vascular intensity, IL-8 expression with moderate vascular intensity, and IL-8 expression with severe vascular intensity were scored as 0, 1, 2, and 3, respectively.
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