Immunofluorescence was carried out to detect the distribution of ZO-1 and occludin proteins in the RPE-choroid complex in all groups. Briefly, fixed tissues were washed 3 times with PBS, covered with 10% normal donkey serum diluted in PBS, and incubated for 20 min at 37°C. Rabbit anti-ZO-1 polyclonal antibody was used at a 1:100 dilution (no. 40–2200; Invitrogen) and mouse anti-occludin monoclonal antibody at a 1:250 dilution (no. 33–1500; Invitrogen). PBS was used as a control for the primary antibody. Following overnight incubation with the primary antibody at 4°C temperature, the slides were rinsed 3 times with PBS and AlexaFluor 488 was added at a dilution of 1:500 for 1 h at 37°C.
Rabbit anti zo 1 polyclonal antibody
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
The Rabbit anti-ZO-1 polyclonal antibody is a laboratory reagent used to detect the Zonula Occludens-1 (ZO-1) protein, a tight junction-associated protein, in biological samples. This antibody can be used in various immunodetection techniques, such as Western blotting and immunohistochemistry, to study the expression and localization of ZO-1 in cells and tissues.
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Lab products found in correlation
Mouse anti occludin monoclonal antibody, by Thermo Fisher Scientific (2 mentions)
Mouse anti claudin 4 monoclonal antibody, by Thermo Fisher Scientific (2 mentions)
Lsm 700, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 conjugated goat anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg antibody, by Abcam (1 mentions)
Mouse monoclonal anti e cadherin antibody, by BD (1 mentions)
Protease inhibitors, by Boehringer Ingelheim (1 mentions)
Enhanced chemiluminescence kit, by GE Healthcare (1 mentions)
Axioskop 40 microscope, by Zeiss (1 mentions)
Axiocam mrm, by Zeiss (1 mentions)
Rabbit anti occludin polyclonal antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Anti β actin mouse monoclonal antibody, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bca protein concentration assay kit, by Beyotime (1 mentions)
Confocal microscope, by Zeiss (1 mentions)
Alexa fluor 568 conjugated donkey anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Goat serum, by Jackson ImmunoResearch (1 mentions)
11 protocols using rabbit anti zo 1 polyclonal antibody
1
Immunofluorescence Analysis of RPE-Choroid Complex
2
Immunofluorescence Imaging of Tight Junction Proteins
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Cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min, and permeabilized with PBS containing 0.5% Triton X-100 and 3% bovine serum albumin (BSA) for 10 min. The cells were then incubated with each primary antibody prepared in PBS containing 1% BSA at 4 °C overnight, followed by incubation with Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (1:500; Abcam) or Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (1:500; Invitrogen) and nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI). Images of the stained cells were obtained with a confocal laser microscope (LSM700; Zeiss, Jena, Germany). The primary antibodies were as follows: rabbit anti-claudin-1 polyclonal antibody (Invitrogen); rabbit anti-claudin-3 polyclonal antibody (Abcam); mouse anti-claudin-4 monoclonal antibody (Invitrogen); rabbit anti-occludin polyclonal antibody (Thermo Fisher Scientific); rabbit anti-ZO-1 polyclonal antibody (Invitrogen); and mouse anti-E-cadherin monoclonal antibody (BD Biosciences).
3
Quantification of Tight Junction Proteins in Guinea Pig RPE-Choroid
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Western blot analysis was perforemd to detect the protein expression of ZO-1 and occludin in the RPE-choroid complex. The RPE-choroid complex of the guinea pig eyes was collected and lysed in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 2 M MEDTA, 1% NP-40) containing protease inhibitors (Boehringer, Mannheim, Germany). Total protein was resolved by SDS polyacrylamide gel electrophoresis, and then was transferred onto a nitrocellulose membrane. The membrane was incubated at 4°C overnight with rabbit anti-ZO-1 polyclonal antibody (1:250 dilution; no. 61–7300; Invitrogen) and mouse anti-occludin monoclonal antibody (1:500 dilution; no. 33–1500; Invitrogen). Peroxidase-conjugated secondary antibodies were used as secondary detection reagents with an enhanced chemiluminescence kit (GE Healthcare, New York, NY, USA). Chemiluminescent signals were visualized by exposure to X-ray film. Band intensities were quantified with BandScan software (version 5.0). Levels of GAPDH were used for standardization. The relative expression of the target protein was calculated. Independent experiments were performed, and repeated 3 times.
4
Immunofluorescence Staining of FFPE Tissue
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Immunofluorescence staining was performed on FFPE tissue sections using several primary antibodies: rabbit anti-occludin polyclonal antibody (1:100, Invitrogen, Darmstadt, Germany), rabbit anti-ZO-1 polyclonal antibody (1:50, Invitrogen), rabbit anti-granzyme B polyclonal antibody (1:100, Abcam, Cambridge, United Kingdom) and goat anti-IgR polyclonal antibody (1:250, R&D systems, Minneapolis, USA) applied by manufacturer instructions. Primary antibodies were visualized by DyLight®594 or DyLight®488 conjugated donkey anti-rabbit polyclonal secondary antibody (1:500, Abcam) and Alexa Fluor 488 conjugated donkey anti-goat polyclonal antibody (1:500, Abcam). Nuclear counterstaining was performed with a mounting medium containing DAPI (Vectashield, Vector Laboratories, USA) and examined using the Zeiss Axioskop 40 microscope (Carl Zeiss Microscopy GmbH, Göttingen, Germany) connected to an AxioCam MRm (Carl Zeiss). Number of granzyme B cells was determined by counting positive cells per 1 cm ileal tissue.
5
Protein Expression and Western Blotting
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RIPA cell lysate in suitable volume was added to the cells. After centrifugation, by the bicinchoninic acid (BCA) protein concentration assay kit (Beyotime, Shanghai, China), we got the concentration of the protein in the supernate. Before being diluted by a 5× Loading Buffer, the samples were boiled with water bath lasting for 5 minutes. Then, we used SDS-PAGE to separate the protein and transferred them to a PVDF membrane (Millipore, USA). Since being incubated with primary antibody, including mouse anti-β-actin monoclonal antibody (diluted 1 : 1000; Cell Signaling Technology), mouse anti-claudin-4 monoclonal antibody (diluted 1 : 500; Invitrogen), rabbit anti-ZO-1 polyclonal antibody (diluted 1 : 250; Invitrogen), rabbit anti-ZONAB polyclonal antibody (diluted 1 : 1000; Invitrogen), and goat secondary antibody conjugated to horseradish peroxidase (diluted 1 : 5000; Sangon, Shanghai, China), ECL luminescence solution (Sigma, St. Louis, MO) was added as luminous substrate, and the band intensity was analyzed by a digital scanning imaging system.
6
Immunostaining of BEST1 and ZO-1 in Cells
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Cells were washed in ice-cold phosphate-buffered saline (PBS) and fixed in 2–4% paraformaldehyde at 4℃ for 30 minutes. Fixed cells were washed twice in PBS and placed in blocking solution (10% normal donkey or goat serum and 0.01–0.05% Triton-X100 in PBS) for 1 hour at room temperature. Cells were then incubated overnight at 4℃ with mouse anti-BEST1 monoclonal antibody (E6-6) (Thermo Fisher Scientific) and rabbit anti-ZO-1 polyclonal antibody (Thermo Fisher Scientific). The following day, cells were washed three to five times in PBS with 0.01% Triton-X100 and incubated with Alexa Fluor 488-conjugated goat anti-rabbit antibody (Thermo Fisher Scientific) and Alexa Fluor 568-conjugated donkey anti-rabbit antibody (Thermo Fisher Scientific). After secondary antibody incubation, cells were stained with DAPI (Thermo Fisher Scientific), washed three times in PBS with 0.2% Triton-X100, and imaged on a confocal microscope (Zeiss, Jena, Germany).
7
Immunofluorescence Analysis of Tight Junction Protein
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Cells were cultured on
coverslips (Fisher Scientific, Ottawa, ON, Canada) for 1 week and
then treated with control, DON (1 μg/mL) or DON detoxified by
SMBS (0.5%) or ES (containing 0.5% of SMBS) for 24 h. After treatments,
the cells were fixed with 4% paraformaldehyde (PFA) (Sigma, Oakville,
ON, Canada). The cells were first blocked with 5% goat serum (Jackson
ImmunoResearch Laboratories, West Grove, PA) for 1 h at room temperature
and then incubated with rabbit anti-ZO-1 polyclonal antibody (1:100
dilution, Thermo Scientific) at 4 °C overnight. The cells were
washed three times with PBS and incubated with Alexa fluor 488 goat
anti-rabbit (Thermo Scientific, Cat. No. A-11034) for 1 h at room
temperature. After three washes with PBS, the cells were mounted with
Vectashield Mounting Medium with DAPI (Vector Laboratories, Burlingame,
CA) and images were taken on a Zeiss fluorescence microscope (Carl
Zeiss Canada Ltd, Toronto, ON, Canada).
coverslips (Fisher Scientific, Ottawa, ON, Canada) for 1 week and
then treated with control, DON (1 μg/mL) or DON detoxified by
SMBS (0.5%) or ES (containing 0.5% of SMBS) for 24 h. After treatments,
the cells were fixed with 4% paraformaldehyde (PFA) (Sigma, Oakville,
ON, Canada). The cells were first blocked with 5% goat serum (Jackson
ImmunoResearch Laboratories, West Grove, PA) for 1 h at room temperature
and then incubated with rabbit anti-ZO-1 polyclonal antibody (1:100
dilution, Thermo Scientific) at 4 °C overnight. The cells were
washed three times with PBS and incubated with Alexa fluor 488 goat
anti-rabbit (Thermo Scientific, Cat. No. A-11034) for 1 h at room
temperature. After three washes with PBS, the cells were mounted with
Vectashield Mounting Medium with DAPI (Vector Laboratories, Burlingame,
CA) and images were taken on a Zeiss fluorescence microscope (Carl
Zeiss Canada Ltd, Toronto, ON, Canada).
8
Visualizing Tight Junction Proteins in Caco-2 Cells
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After 15 days of differentiation, Caco-2 on glass coverslips were incubated with 2 and 1mg/mL A. simplex CE and fixed in 3% formaldehyde at two and 24 hours. Thereafter, the Caco-2 cells were permeabilized with 0.1% TX-100, 0.2% BSA in PBS for 10 minutes and then blocked with 2% BSA in PBS for one hour. Subsequently, the cells were incubated with rabbit anti-ZO-1 polyclonal antibody (40–2200, 1:100, Thermo Fisher Scientific) and mouse anti-occludin monoclonal antibody (OC-3F10, 1:200, Thermo Fisher Scientific) for 90 minutes. Then, the cells were incubated with Alexa Fluor-488-labelled goat-anti-rabbit IgG (H+L) (A-11008, 1:1,000, Thermo Fisher Scientific) and Alexa Fluor-647-labelled goat anti-mouse IgG (H+L) (A-21235, 1:500, Thermo Fisher Scientific) as secondary antibodies for one hour. Localization and distribution of TJ proteins were analysed with a Leica TCS SP5 confocal microscope (Mannheim, Germany) with an AOBS (Acousto-Optical Beam Splitter) with 40X and 63X oil immersion optics. Laser lines at 488nm and 633nm were provided by an Argon laser and a HeNe laser. Detection ranges were set to eliminate crosstalk between fluorophores. Four experiments were performed. Assays were performed in the Confocal Laser and Multidimensional Microscopy in vivo Service of CIB-CSIC.
9
Immunofluorescence Imaging of Tight Junctions
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CFS cells were harvested from the monolayer culture using trypsin and seeded onto a 96-well plate at 1 × 10 4 cells/well and incubated at 20 • C until the cells formed a monolayer. The wells were then treated with anti-cTecrem mAb, anti-SCR1-2 pAb, and anti-SCR3-4 pAb at 25 • C for 48 h. The cells were washed with PBS, fixed with 200 µL of methanol. After washing twice with PBS, the wells were blocked by incubating with 1% BSA in 0.05% Tween 20-PBS for 1 h at room temperature. The cells were incubated with a 5 µg/mL concentration of rabbit anti-ZO1 polyclonal antibody (#61-7300, ThermoFisher Scientific, USA) for 1 h at room temperature, followed by 3x PBST and 2x PBS washes. After secondary antibody (anti-rabbit IgG-FITC) treatment and washings with PBST and PBS, the cells were counterstained with 1 µg/mL DAPI for 10 min and observed under the fluorescent microscope. The images were taken with a Leica DFC300F camera, and the fluorescent intensity of the cells were measured using ImageJ software.
10
Immunoblot Assay Protocol Compendium
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Immunoblot assay were performed as described previously [25] (link), [26] (link). The following antibodies were used as primary antibodies: polyclonal rabbit anti-VASH1 antibody [32] (link); polyclonal rabbit anti-TGF-β1/2/3 antibody (Santa Cruz Biotechnology); monoclonal rabbit anti-phosphorylated Smad3 (pSmad3) antibody and monoclonal rabbit anti-Smad3 antibody (Cell Signaling Technology); polyclonal rabbit anti-VEGF-A antibody (Santa Cruz Biotechnology); polyclonal rabbit anti-Angiopoietin-1 (Ang-1) antibody and polyclonal rabbit anti-Angiopoietin-2 (Ang-2) antibody (Alpha Diagnostic, San Antonio, TX); polyclonal rabbit anti-IκBα antibody (Santa Cruz Biotechnology); monoclonal rabbit anti-phosphorylated IκBα (pIκBα) antibody (Cell Signaling Technology); polyclonal rabbit anti-ZO-1 antibody (Invitrogen) and polyclonal rabbit anti-beta actin antibody (Abcam).
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