The experiments were performed on the in vitro model of BBB consisting of a mixed culture of endothelial, astroglial and neural cells. Isolation and preparation of the primary brain microvessels (BMV) culture were performed according to the protocol of Liu et al. [55 (link)]. The BMVs were phenotyped with monoclonal antibodies to the endothelial marker the zonula occludens-1 (ZO1) using a standard immunohistochemistry protocol using primary anti-ZO1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and secondary antibodies labeled with Alexa Fluor 488 (Abcam, Cambridge, United Kingdom) followed by an Olympus FV10i-W confocal laser scanning microscope (Olympus, Tokyo, Japan). Astroglial and neural cells for the in vitro BBB model are derived from embryonic neurospheres as described in Ref. [56 ]. The TEER was measured using an EVOM2 volt-ohmmeter with the STX-2 electrodes (World Precision Instruments, Sarasota, FL, USA). Resistance values (Ωcm2) were corrected by subtracting the resistance of an empty Transwell filter.
Anti zo 1 antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Anti-ZO-1 antibody is a research-use antibody targeting the Zonula Occludens-1 (ZO-1) protein. ZO-1 is a tight junction-associated protein involved in the regulation of epithelial and endothelial barrier function. This antibody can be used for the detection and analysis of ZO-1 in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Abcam (2 mentions)
Anti occludin antibody, by Santa Cruz Biotechnology (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Fv10i w confocal laser scanning microscope, by Olympus (1 mentions)
Stx2 electrode, by World Precision Instruments (1 mentions)
Evom2 voltohmmeter, by World Precision Instruments (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Anti occludin, by Santa Cruz Biotechnology (1 mentions)
Anti claudin 5, by Santa Cruz Biotechnology (1 mentions)
Anti cysteine sulfenic acid antibody, by Merck Group (1 mentions)
Anti p p38 antibody, by Cell Signaling Technology (1 mentions)
Dimedone, by Tokyo Chemical Industry (1 mentions)
Alexa 680 fluor c2 maleimide, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated anti rabbit or mouse igg, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Biotinylated goat anti rabbit immunoglobulin g igg, by Vector Laboratories (1 mentions)
Avidin biotin complex kit, by Vector Laboratories (1 mentions)
Biotinylated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
13 protocols using anti zo 1 antibody
1
In vitro Blood-Brain Barrier Model
2
Western Blot Analysis of Tight Junction Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell suspensions were prepared from dissected ipsilateral cortices and transferred to a fresh tube. The suspensions were homogenized using a handheld mortar and pestle and then agitated for 10 min. Extracts were clarified by centrifugation and then diluted in a reducing agent. Proteins were resolved on a 12% Bis-Tris polyacrylamide gel and electrotransferred onto a nitrocellulose membrane. The membrane was incubated with anti-claudin-5, anti-occludin or anti-ZO-1 antibodies (1:200; Santa Cruz Biotechnology, Inc.), followed by incubation with goat-anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:1,000; Santa Cruz Biotechnology, Inc.) in blocking buffer. The membrane was then developed using enhanced chemiluminescence detection and film exposure (Amersham, Little Chalfont, UK). GAPDH was used as the internal control.
3
Detecting Cysteine Sulfenic Acid Modifications
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bovine serum albumin (BSA, Faction V) was obtained from Iwai Chemical Company (Tokyo, Japan). Dimedone was purchased from Tokyo Chemical Industry (Tokyo, Japan), Anti-cysteine sulfenic acid antibody was from Millipore (MA, USA). HRP-conjugated anti-rabbit or mouse IgG, anti-β-actin and anti-p-P38 antibodies were purchased from Cell Signaling, Inc. (Beverly, MA). Anti-E-Cadherin and anti-ZO-1 antibodies were bought from Santa Cruz Biotechnology (Santa Cruz, CA). Alexa 680 Fluor C2 Maleimide was from Thermo Scientific (Rockford, IL). Sodium dextran sulfate 5000 (DSS) was from Wako Pure Chemical Corporation (Japan). Maleimide and all other chemicals were from Sigma-Aldrich (St. Louis, MO).
4
Immunohistochemical Analysis of Tight Junction Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After routine depara nization and rehydration, heat-induced epitope retrieval was performed through the immersion of slides in 0.01 M sodium citrate buffer (pH 6.0). To block endogenous peroxidase activity and nonspeci c antibody binding, the sections were rst preincubated in 0.1 M phosphate buffered saline (PBS) containing 10% normal goat serum and 0.3% H 2 O 2 for 1 h at room temperature before incubation with rabbit polycolonal anti-ZO-1 antibodies (1:50; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and mouse monocolonal antioccludin (1:50 dilution; Santa Cruz Biotechnology) as primary antibodies for 20 h at 4°C. The sections were then treated for 1 h at 37°C with biotinylated goat antirabbit immunoglobulin G (IgG; 1:200, Vector Laboratories, Burlingame, California, USA) for anti-ZO-1 antibodies and with biotinylated goat antimouse IgG (1:200; Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, USA) for antioccludin antibodies. Sections were subsequently reacted with reagents from an avidin-biotin complex kit (Vector Laboratories), and the brown reaction products were visualized following the use of a diaminobenzidine substrate kit (Vector Laboratories) per the manufacturer's recommendations.
5
Establishing an In Vitro BBB Model
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell culture models, based on immortalized brain endothelial cell lines, have been developed in order to facilitate in vitro studies of drug transport to the brain [25] (link). The best the BBB models in vitro which mimic the in vivo BBB integrity are characterized by high transendothelial electrical resistance (TEER regularity above 200 Ohm × cm (2)) and the expression of tight junction (TJ) proteins as critical factors of the BBB physiology and structure [26] .
Brain microvessel endothelial cells (BMECs) were isolated from the brain of Wistar rats (postnatal day 10-14). Isolation and establishment of primary culture of BMECs were done according to the protocol of Liu et al. [27] .The BMECs obtained were phenotyped by the standard immunohistochemistry protocol using primary anti-ZO1 antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX, USA, sc-8147) and secondary antibodies labeled with Alexa Fluor 488 (Abcam, Cambridge, UK, ab150117) followed by the detection with the fluorescent microscope. Astroglial and neuronal cells for the BBB model were obtained from embryonic rat neurospheres as described in [28] . The integrity of the in vitro BBB model containing BMECs monolayer grown on inserts, astrocytes and neurons grown in the well was confirmed with the TEER measurements.
Brain microvessel endothelial cells (BMECs) were isolated from the brain of Wistar rats (postnatal day 10-14). Isolation and establishment of primary culture of BMECs were done according to the protocol of Liu et al. [27] .The BMECs obtained were phenotyped by the standard immunohistochemistry protocol using primary anti-ZO1 antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX, USA, sc-8147) and secondary antibodies labeled with Alexa Fluor 488 (Abcam, Cambridge, UK, ab150117) followed by the detection with the fluorescent microscope. Astroglial and neuronal cells for the BBB model were obtained from embryonic rat neurospheres as described in [28] . The integrity of the in vitro BBB model containing BMECs monolayer grown on inserts, astrocytes and neurons grown in the well was confirmed with the TEER measurements.
6
Quantitative Immunoblotting of Tight Junction Proteins
7
Immunofluorescence Analysis of Cell Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The mouse antibody anti-H3K9me2/3 (#5327), and the rabbit antibody -phospho-ERM (#3726) were from Cell Signaling (Danvers, MA, USA). The mouse antibody anti-β-tubulin (#T5201) was from Sigma-Aldrich. The antibody anti-α4 (HP2/1) was a gift from Prof. Sánchez-Madrid (Hospital de la Princesa, Universidad Autónoma de Madrid). The anti-ZO-1 antibody was from SantaCruz (Santa Cruz, CA, USA) (#sc-33725). Tetramethylrhodamine (TRITC)-Phalloidin, Alexa 647-Phalloidin, CellTrace™ CFSE, secondary antibodies Alexa-488, -594, -647 for immunofluorescence analysis and DAPI were obtained from Thermo Scientific (Waltham, MA, USA). VCAM1 was obtained from Peprotech. BIX01294 was from Abcam (Cambridge, UK).
8
Analyzing Retinal Cryosections and Flat-Mounts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For cryosections, eyes were removed carefully and embedded in optimal cutting temperature compound (Sakura Fine Technical). The frozen samples were then sliced transversely (8 μm) with a cryostat (Leica CM1950) at –20 °C. Only the cross-sections throughout optic nerve were used for staining and analysis. For retinal-RPE flat-mount, the eyes were fixed in 4% PFA for 60 min at room temperature and the RPE-sclera complex were dissected out. The cryosection, RPE flat-mount sheet samples and ARPE cells were blocked with 0.5% Triton-X100/5% BSA and incubated with primary antibodies overnight at 4 °C. The primary antibodies included anti-ZO-1 antibody (Santa Cruz, sc-33725), anti-Arg1 antibody (Santa Cruz, sc-18355), anti-Nos2 antibody (Santa Cruz, sc-7271), anti-IL-4Ra antibody (Santa Cruz, sc-28361), anti-IL-4 antibody (Santa Cruz, sc-53084), and anti-p-Nrf2 antibody (Abcam, ab76026). Then they were incubated with secondary antibodies for 1 h and counterstained with DAPI (Invitrogen, D1306) for 5 min before mounted. Negative control was done by omission of the primary antibody. The images were obtained using a confocal microscope (LSM880, Carl Zeiss).
9
Identification of Hippocampal Microvascular Endothelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD31ZO-1 double positive hippocampus microvascular endothelial cells were identified using an anti-CD31 antibody (eBioscience, San Diego, USA) and an anti-ZO1 antibody (Santa Cruz Biotechnology). Cell surface staining of freshly isolated brain single cell suspensions was performed with the fluorescently labelled antibodies at 4°C for 30 minutes. All flow cytometry measurements were carried out using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). Analysis was performed using CellQuestTM software, version 3.1 (Becton Dickinson).
10
Immunofluorescence Analysis of Murine Colon
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen sections of mice colon tissues were first fixed in acetone and permeabilized, and then blocked with 5% normal goat serum at room temperature for 1 h before incubating with primary antibody followed by secondary antibody. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) working solution. The fluorescent staining of colonic tissue was observed using a fluorescent microscope (Nikon) and images were obtained using Case Viewer software (version 2.3, 3D Histech). The average intensity of immunofluorescence staining was analyzed semi-quantitatively using ImageJ software. The primary antibodies were used as follows: anti-ZO-1 antibody (SantaCruz Biotechnology), anti-occludin antibody (SantaCruz Biotechnology), anti-F4/80 antibody (Cell Signaling Technology), anti-CD86 antibody (Cell Signaling Technology), anti-CD206 antibody (Cell Signaling Technology) and anti-integrin β6 antibody (Cell Signaling Technology).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!