GF mouse ileum and colon tissues were removed, washed with PBS, and fixed in 4% paraformaldehyde, dehydrated, cleared, and embedded in paraffin. The paraffin sections were deparaffinized, rehydrated, and treated with EDTA antigen retrieval buffers. The slides were immunostained with anti-ZO-1 (1:100, Proteintech Group) and anti-occludin (1:50, Proteintech Group) antibodies overnight at 4°C, followed by incubation with a goat anti-rabbit Cy3-conjugated secondary antibody (1:200, Servicebio) for 1 h in total darkness. DAPI was used for nuclei staining. Fluorescence images were captured using a Nikon Eclipse Ti-SR fluorescence microscope at a magnification of ×20.
Anti zo 1
Manufactured by Proteintech
Sourced in United States, China, United Kingdom
Anti-ZO-1 is a primary antibody that recognizes the Zonula Occludens-1 (ZO-1) protein. ZO-1 is a tight junction-associated protein that plays a crucial role in the regulation of epithelial and endothelial cell-cell adhesion and barrier function. This antibody can be used to detect and study the expression and localization of ZO-1 in various cell and tissue types.
Automatically generated - may contain errors
Lab products found in correlation
Anti occludin, by Proteintech (10 mentions)
Anti e cadherin, by Proteintech (5 mentions)
Anti claudin 1, by Proteintech (3 mentions)
Anti cd31, by Abcam (3 mentions)
Anti e cadherin, by Cell Signaling Technology (3 mentions)
Anti gapdh, by Proteintech (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Anti caspase 3, by Cell Signaling Technology (2 mentions)
Anti β actin, by Proteintech (2 mentions)
Anti claudin 5, by Thermo Fisher Scientific (2 mentions)
Anti n cadherin, by Cell Signaling Technology (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Cy3 conjugated goat anti rabbit secondary antibody, by Wuhan Servicebio Technology (1 mentions)
Eclipse ti sr fluorescence microscope, by Nikon (1 mentions)
Anti cd31, by Proteintech (1 mentions)
Anti cyp3a4, by Proteintech (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Proteintech (1 mentions)
Triton x 100, by Merck Group (1 mentions)
33 protocols using anti zo 1
1
Immunofluorescent Localization of Tight Junction Proteins
2
Immunofluorescence Staining of Cultured Tissue
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After 7 days of culture, the device was disassembled, and the cultured tissue was fixed in 4% paraformaldehyde for 30 min at room temperature and then permeabilized with 0.2% Triton X-100 (Sigma) in PBS solution for 15 min at room temperature. After blocking with 2% bovine serum albumin (BSA, Sigma) in PBS for 1 h at room temperature, samples were incubated overnight at 4 °C with primary antibodies (anti-CD31, anti-ZO-1, anti-CYP1A2, and anti-CYP3A4; 1:100 in PBS; Proteintech Co., Ltd., Hubei, China) and then incubated for 4 h at 4 °C with the corresponding secondary antibodies (Alexa Fluor 488 or 594; 1:250 in PBS; ABclonal Biotech Co., Ltd., Hubei, China). Cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; 1:100 in PBS; Proteintech Co., Ltd., Hubei, China) for 30 min at room temperature. The samples were washed 3 times with PBS after each step (Fig. S14B and Fig. S14C ). Images of the samples were taken using CLSM.
3
Protein Extraction and Western Blotting
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Total protein was extracted from frozen lung tissues using RIPA and phenylmethylsulfonyl fluoride (PMSF) (99:1) (Beyotime, Shanghai, China). Protein concentrations were determined using the BCA kit (Beyotime). Protein extracts were subjected to SDS-PAGE and visualized with SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher, MA, United States). The following primary antibodies were purchased from Proteintech and used for western blotting: anti-GAPDH (1:5,000), anti-AGR3 (1:500), anti-E-cadherin (1:8,000), anti-occludin (1:5,000), and anti-ZO-1 (1:5,000).
4
Intestinal Protein Expression Analysis
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Intestine tissue (n = 4 pups/group) soaked in RIPA lysis buffer (Beyotime, China) supplemented with protease inhibitor (Beyotime, China) was homogenized using an electric homogenizer and centrifuged to obtain the supernatant. Protein concentrations were measured using a Pierce BCA Protein Assay Kit (Beyotime, China). The protein supernatant was mixed with sodium dodecyl sulfate sample buffer (Beyotime, China) at a ratio of 4:1 and denatured at 100 ℃ for 10 min. Protein samples were separated in 10% polyacrylamide gels and transferred to 0.45 μm PVDF membranes, and measured using anti-TLR4, anti-NF-κB P65 (Servicebio, China), anti-ZO-1, anti-Occludin, anti-Claudin-1 (Proteintech, China) and β-Actin (ZENBIO Biotechnology, China) at 4 ℃ overnight. Signals were detected using chemiluminescence (ECL Western Blotting Substrate, Bio-Rad). The relative intensity of target bands was quantified using the Image J analysis system (Bio-Rad).
5
Evaluating Cancer Stem Cell Properties
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The Cell-Light 5-ethynyl-2′-deoxyuridine (EdU) Apollo 488 in vitro kit (C10310-3) was purchased from RiboBio (Guangzhou, China). Oxaliplatin (OXA) was purchased from the Cancer Hospital of the Chinese Academy of Medical Sciences (Beijing, China). MTT was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-GAPDH, anti-NANOG, anti-Snai1, anti-p38, and anti-p-p38 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-OCT4, anti-SOX2, anti-ZO1, anti-E-cadherin, anti-CD133, and anti-Ki67 antibodies were purchased from Proteintech Group, Inc. (Rosemont, IL, USA). Anti-S100A8 antibodies were purchased from Beyotime (Nantong, China).
6
Investigating Cell Death Mechanisms
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Minocycline hydrochloride, Nec-1s, GSK-872, and Z-VAD-FMK were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Anti-HMGB1 antibody and anti-rabbit IgG (H+L), F(ab')2 fragment (Alexa Fluor 488 Conjugate) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-ZO-1, anti-RIPK3 and anti-MLKL antibodies were purchased from Proteintech (Rosemont, IL, USA). Mitochondrial membrane potential assay kit with JC-1 and ROS assay kit were purchased from Beyotime Biotechnology (Shanghai, China). DMEM/F12 and DMSO were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum was purchased from Gibco (Logan, UT, USA). Dead cell apoptosis kit with Annexin V Alexa Fluor 488 & Propidium Iodide (PI), goat anti-rabbit IgG (H+L) (Alexa Fluor 488), and goat anti-mouse IgG (H+L) (Alexa Fluor 568) were purchased from Invitrogen (Carlsbad, CA, USA).
7
Histological Analysis of Liver and Colon Damage
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Appropriately sized samples were obtained from the left liver and colon and immediately fixed in 10% formalin for 24 h. Then, paraffin was used to embed the tissue. H&E staining and a histological activity index analysis were performed to estimate liver damage (Knodell et al., 1981 ).
The liver samples were cut into 4‐µm‐thick sections and stained with anti‐phospho‐p65, anti‐Ly6G, or anti‐F4/80 antibodies. The colon samples were cut and stained with anti‐ZO‐1 (Proteintech, Rosemont, IL, USA) antibodies according to a previously reported immunofluorescence protocol (Chung et al.,2014 ). The images were visualized by P250 FLASH (3D HISTECH, Budapest, Hungary).
The liver samples were cut into 4‐µm‐thick sections and stained with anti‐phospho‐p65, anti‐Ly6G, or anti‐F4/80 antibodies. The colon samples were cut and stained with anti‐ZO‐1 (Proteintech, Rosemont, IL, USA) antibodies according to a previously reported immunofluorescence protocol (Chung et al.,
8
Immunofluorescence Staining Visualization
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For immunofluorescence detection, sections and cells were incubated with primary antibody and then labeled with an anti-rabbit secondary antibody conjugated with Cy3 or FITC (1:1000) in the dark for 1 h at room temperature. Cell nuclei were counterstained with 40, 60-diamidino-2-phenylindole (DAPI). Images were acquired under a laser scanning confocal microscope (Carl Zeiss Jena) or fluorescence inverted microscope (Nikon). The following antibodies were used: anti-caspase-3 (1:2000, Cell Signaling Technology), anti-ZO-1 (1:1000, Proteintech), anti-Oculdin-3 (1:2000, Proteintech), anti-γ-H2AX (1:1000, Cell Signaling Technology), and anti-Ki-67 (1:1000, Cell Signaling Technology).
9
Western Blot Analysis of Tight Junction Proteins
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Cells were rinsed with ice-cold PBS buffer thrice and lysed with RIPA lysis buffercontaining 1 mM PMSF on ice for 30 min. The cell debris was subsequently removed by centrifugation at 12000g for 10 min at 4 °C. Then the supernatant was collected for analysis by adding using 5 × SDS loading buffer containing 7% β-mercaptoethanol. Equal amounts of protein samples were separated by 10% (v/v) SDS-PAGE and transferred onto PVDF membranes (0.22 μm, Millipore, USA). After blocking with 5% BSA at room temperature for 2 h, the membranes were incubated with the corresponding primary anti-ZO-1 (1:1000; Proteintech, USA), anti-GAPDH (1:8000; Sigma, USA) and anti-Occludin (1:1000; Proteintech, USA) antibodies at 4 °C overnight. The membranes were then incubated with HRP-conjugated anti-rabbit IgG (1:4000; Cell Signaling Technology, USA) secondary antibodies for 2 h at room temperature, and the immunoreactive protein bands were visualized using ECL detection reagents (Bio-Rad, USA). The intensity of protein bands was quantitated using an Image Lab analysis software and normalized to GAPDH.
10
Immunofluorescence Staining of Endothelial Markers
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Sections were fixed for 15 minutes with 4% paraformaldehyde, permeabilized with Triton X‐100 (0.1%), and blocked with solution containing 5% bovine serum before applying primary antibody.
Specimens were incubated, respectively, with anti‐CD31 (catalog no. ab24590; 1:200; Abcam, Cambridge, MA); anti–prospero homeobox 1 (catalog no. 11067‐2‐AP; 1:100; ProteinTech Group Inc, Rosemont, IL); anti–vascular endothelial/cadherin (VE‐cadherin; catalog no. ab33168; 1:300; Abcam); anti–zonula occludens‐1 (ZO‐1; (catalog no. 21773‐I‐AP; 1:100; ProteinTech Group); anti‐ZO‐1 (catalog no. 66452‐1‐lg; 1:100; ProteinTech Group); anti–collagen IV (catalog no. ab19808; 1:200; Abcam); and anti‐fibroblast‐specific factor1 (catalog no. S100A4; 1:200; Abcam) for 12 hours in 4°C. Secondary antibodies (catalog no. A21207; 1:200; Life Technologies, Carlsbad, CA) and Alexa Fluor 488 goat antimouse immunoglobulin G (catalog no. A11001; Life Technologies) were incubated subsequently under light‐protected conditions for 1 hour at room temperature. Nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (catalog # KGA215–10; KeyGEN BioTECH Corp., Ltd, Nanjing, China) in the end. After final washing, coverslips were mounted on slides using 50% glycerin. Then, sections were observed under a fluorescence microscope (Olympus, Tokyo, Japan) or confocal laser scanning microscope (Olympus).
Specimens were incubated, respectively, with anti‐CD31 (catalog no. ab24590; 1:200; Abcam, Cambridge, MA); anti–prospero homeobox 1 (catalog no. 11067‐2‐AP; 1:100; ProteinTech Group Inc, Rosemont, IL); anti–vascular endothelial/cadherin (VE‐cadherin; catalog no. ab33168; 1:300; Abcam); anti–zonula occludens‐1 (ZO‐1; (catalog no. 21773‐I‐AP; 1:100; ProteinTech Group); anti‐ZO‐1 (catalog no. 66452‐1‐lg; 1:100; ProteinTech Group); anti–collagen IV (catalog no. ab19808; 1:200; Abcam); and anti‐fibroblast‐specific factor1 (catalog no. S100A4; 1:200; Abcam) for 12 hours in 4°C. Secondary antibodies (catalog no. A21207; 1:200; Life Technologies, Carlsbad, CA) and Alexa Fluor 488 goat antimouse immunoglobulin G (catalog no. A11001; Life Technologies) were incubated subsequently under light‐protected conditions for 1 hour at room temperature. Nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (catalog # KGA215–10; KeyGEN BioTECH Corp., Ltd, Nanjing, China) in the end. After final washing, coverslips were mounted on slides using 50% glycerin. Then, sections were observed under a fluorescence microscope (Olympus, Tokyo, Japan) or confocal laser scanning microscope (Olympus).
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