Proteins from exosomes and BMECs were extracted, and the concentration was determined using a BCA assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). The resolved proteins were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The proteins were blocked in 5% milk and incubated with primary antibodies against CD63 (1:1000, EXOAB-CD63A-1, SBI), ZO-1 (1:1000, 13663S, Cell Signaling Technology), claudin-5 (1:1000, Ab131259, Abcam), N-cadherin (1:5000, Ab76011, Abcam), Occludin (1:1000, Ab216327, Abcam) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 60004-1-Lg, Proteintech) overnight at 4°C. The samples were then incubated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies. Enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used to show the protein bands, and optical density was assessed via ImageJ software.
Ab131259
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab131259 is a primary antibody product manufactured by Abcam. It is designed for use in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab216327, by Abcam (9 mentions)
Ab8245, by Abcam (6 mentions)
Ab216880, by Abcam (5 mentions)
Ab33168, by Abcam (4 mentions)
Ab8226, by Abcam (4 mentions)
Ab32570, by Abcam (3 mentions)
Ab7871, by Abcam (3 mentions)
Ab236127, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Ab1791, by Abcam (2 mentions)
Sa00001 1, by Proteintech (2 mentions)
Sa00001 2, by Proteintech (2 mentions)
Ab190085, by Abcam (2 mentions)
Ab177487, by Abcam (2 mentions)
Chemiluminescent reagent, by Merck Group (2 mentions)
Gb11132, by Wuhan Servicebio Technology (2 mentions)
Horseradish peroxidase conjugated anti mouse or anti rabbit igg, by Cell Signaling Technology (2 mentions)
Bicinchoninic acid assay kit, by Beyotime (2 mentions)
28 protocols using ab131259
1
Protein Expression Analysis of Exosomes and BMECs
2
Quantification of Tissue Perforin Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The abundance of perforin in the tissues was detected by western blot. In short, the total proteins in the tissues were extracted by RIPA lysis buffer (Beijing Nobleide Technology Co., Ltd., Beijing, China). The total proteins were quantified and then separated with SDS-polyacrylamide gel. After that, the proteins were transferred to polyvinylidene fluoride membranes by wet-transfer methods. Subsequently, the membranes were blocked with 5% fat-free milk for 2 hours and then were incubated with the primary antibodies including anti-perforin (ab256453, Abcam), Occludin (ab216327, Abcam), Claudin-5 (ab131259, Abcam), and anti-β-actin (ab179467, Abcam) at 4°C overnight. The membranes were washed with the TBST for three times and then were incubated with secondary antibodies for 2 hours. Finally, the proteins were quantified by a chemiluminescence detection system.
3
Western Blot Analysis of Cell Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After adjusting the total protein concentration of the cell samples, 50 μg of protein sample was uploaded per well and separated by polyacrylamide gel electrophoresis. The proteins were transferred to PVDF membrane (cat. no. FFP24; Beyotime) and then washed with PBS (cat. no. C0221A; Beyotime) for three times. After 10 min each, the membrane was blocked with 5% skim milk (cat. no. P0216; Beyotime) at room temperature for 2 h. Next, the membrane was probed with the corresponding primary antibodies at 4°C for incubation overnight and then incubated with secondary antibody Goat Anti‐Rabbit IgG H&L (HRP; ab6721; dilution, 1:2000; Abcam) at 37°C for 2 h. After washing with TBST again, it was placed into Odyssey (LI‐COR) for scanning and analysis. The primary antibodies included P2Y12R (ab233760; dilution, 1:1000; Abcam), NF‐κB p65 (ab32536; dilution, 1:1000; Abcam), P‐IKB‐α (ab133462; dilution, 1:10,000; Abcam), IKB‐α (ab32518; dilution, 1:1000; Abcam), p‐AKT (ab38449; dilution, 1:1000; Abcam), eNOS (ab300071; dilution, 1:1000; Abcam), VE‐cadherin (#2500; dilution, 1:1000; Cell Signaling Technology), ZO‐1 (ab216880; dilution, 1:1000; Abcam), Claudin 5 (ab131259; dilution, 1:1000; Abcam), Occludin (ab216327; dilution, 1:1000; Abcam), H3 (ab1791; dilution, 1:1000; Abcam), and GAPDH (ab8245; dilution, 1:1000; Abcam).
4
Western Blot Analysis of Tight Junctions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis was performed as previously reported,30 (link) with minor modifications. In brief, HRMECs were treated with 10-nM SZN-413-p 24 hours before harvesting for cell lysis. The anti-ZO-1 (ab216880; Abcam, Cambridge, UK), anti-CLDN5 (ab131259; Abcam), and anti-β actin (A5441; Sigma-Aldrich, St. Louis, MO) primary antibodies were used, followed by horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies for chemiluminescent signals.
5
Immunoblot Analysis of Inflammatory Mediators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We extracted and denatured the total protein in each group of cells and tissues. After SDS-gelelectrophoresis, we transferred them to nitrocellulose membranes. Then, we incubated the different primary antibodies including TLR4 (1:500, ab13556, Abcam, UK), NLRP3 (1:1000, ab263899, Abcam), IL-1β (1:1000, ab234437, Abcam), IL-18 (1:4000, 60,070-1-Ig, Proteintech, USA), caspase-1 (1:1000, 22,915-1-AP, Proteintech), NF-κB (1:2000, 10,745-1-AP, Proteintech), ASC (1ug/mL, ab175449, Abcam), Pro-MMP9 (2ug/mL, MAB9111, R&D), Claudin5 (1:4000, ab131259, Abcam) and β-actin(1:5000, 66,009-1-Ig, Proteintech)overnight 4 °C.After washing the membranes three times, the blots were incubated with fluorescent-basedanti-rabbit(1:6000, SA00001-2, Proteintech), anti-mouse(1:5000, SA00001-1, Proteintech) or anti-goat(1:5000, SA00001-4, Proteintech) IgG secondary antibody at room temperature.We visualized the proteins by chemiluminescence detection reagent (Thermo Fisher, Waltham, MA, USA). We used Image J software to analyze the gray value of all bands quantitatively.
6
Immunohistochemistry and Blotting Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies against ZO-1 (Abcam, ab190085; dilution 1:1,000), 5hmC (Active Motif, #39769, dilution 1:1,000), GFAP (Abcam, ab7260, dilution 1:1000), NeuN (Abcam, ab177487, dilution 1:1000), CD68 (Abcam, ab237988, 1:1000) and Claudin-5 (Abcam, ab131259, dilution 1:500) were used for immunohistochemistry staining. Antibody against 5hmC (Active Motif, #39769, dilution 1:5,000) was used for dot-blotting. Antibody against ZO-1 (Proteintech, 22601-1-AP, dilution 1:1000) and TET2 (Proteintech, 21207-1-AP, dilution 1:1000) were used for western-blotting.
Hydrogen peroxide solution (H2O2) (Sigma, 323381), N-acetyl cysteine (NAC) (Sigma, A7250), and DAPI (Sigma, D9542) were commercially obtained.
Fluorescein isothiocyanate (FITC)-conjugated dextran (40 kDa) and FITC-conjugated dextran 10 kDa were purchased from Thermo Fisher Scientific Inc. (Waltham, MA USA).
Hydrogen peroxide solution (H2O2) (Sigma, 323381), N-acetyl cysteine (NAC) (Sigma, A7250), and DAPI (Sigma, D9542) were commercially obtained.
Fluorescein isothiocyanate (FITC)-conjugated dextran (40 kDa) and FITC-conjugated dextran 10 kDa were purchased from Thermo Fisher Scientific Inc. (Waltham, MA USA).
7
Histological and Immunofluorescence Analysis of Brain Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For hematoxylin and eosin (H&E) staining, coronal sections of brain tissues, 20 µm thick, were stained using H&E, with incubation of 2 min for hematoxylin and 1 min for eosin. Sections underwent dehydration and permeabilization before microscopic observation (BX63, Olympus, Japan) (Li et al. 2020b (link)).
For immunofluorescence assay, fixed cells or tissue sections were treated to allow penetration of primary antibodies targeting ZO-1 (ab307799, Abcam, UK), Occludin (ab216327, Abcam, UK), Claudin-5 (ab131259, Abcam, UK), and CD31 (Sc-376764, SANTA CRUZ, US), followed by incubation with Alexa Fluor-conjugated secondary antibodies and DAPI staining. Confocal microscopy facilitated the visualization of these markers (Li et al. 2019 (link)).
The immunohistochemistry protocol entailed the use of antibodies specific to NeuN (ab177487, Abcam), TNF-α (ab307164, Abcam), and IL-1β (ab283818, Abcam). Following secondary antibody application and SABC amplification, the DAB chromogen revealed the localization of target proteins, which was counterstained with hematoxylin. Observations were made under an upright microscope (BX63, Olympus, Japan) (Li et al. 2020b (link)).
For immunofluorescence assay, fixed cells or tissue sections were treated to allow penetration of primary antibodies targeting ZO-1 (ab307799, Abcam, UK), Occludin (ab216327, Abcam, UK), Claudin-5 (ab131259, Abcam, UK), and CD31 (Sc-376764, SANTA CRUZ, US), followed by incubation with Alexa Fluor-conjugated secondary antibodies and DAPI staining. Confocal microscopy facilitated the visualization of these markers (Li et al. 2019 (link)).
The immunohistochemistry protocol entailed the use of antibodies specific to NeuN (ab177487, Abcam), TNF-α (ab307164, Abcam), and IL-1β (ab283818, Abcam). Following secondary antibody application and SABC amplification, the DAB chromogen revealed the localization of target proteins, which was counterstained with hematoxylin. Observations were made under an upright microscope (BX63, Olympus, Japan) (Li et al. 2020b (link)).
8
Immunostaining of Endothelial Tight Junctions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HBMECs grown on collagen-coated coverslips in 24-well culture plates were washed in PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. Fixed cells were blocked with 5% BSA and permeabilized with 0.1% Triton X-100 for 30 min at room temperature. HBMECs were then immunostained with VE-cadherin (1:200, ab33168, Abcam), claudin-5 (1:100, ab131259, Abcam), ZO-1 (1:50, 40–2200, Thermo Fisher) or occludin (1:100, ab216327, Abcam) diluted in blocking buffer overnight at 4 °C and washed three times with PBS at 5-min intervals. F-actin staining was performed by using Rhodamine-Phalloidin (100 nmol/L, PHDR1, Cytoskeleton, Denver, CO, USA) at room temperature for 1 h. HBMECs were then incubated with Alexa488-anti-rabbit or Alexa488-anti-mouse secondary antibodies (Molecular Probes, Carlsbad, CA, USA) for 1 h followed by counterstaining the nuclear with Hoechst 33342 (1:10000, H1399, Thermo Fisher) for 15 min at room temperature. Images were captured by confocal microscopy using a Leica TCS SP8 (Leica Microsystems, Buffalo Grove, IL, USA).
9
Western Blot Analysis of Mouse Hippocampus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein in the mouse hippocampus was extracted with an ice-cold RIPA buffer containing a cocktail of protease inhibitors. Protein concentration was measured by a BCA assay kit. Protein samples were separated in 10–15% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (Millipore). Membranes were blocked with 5% milk in TBS with 0.1% (v/v) Tween-20 (TBST) for 2 h at room temperature and incubated overnight at 4°C with primary antibodies. The primary antibodies applied are as follows: anti-vascular endothelial (VE)-Cadherin (ab205336, 1:1,000, Abcam), anti-zonula occludens-1 (ZO-1, Ab61357, 1:1,000, Abcam), anti-glial fibrillary acidic protein (GFAP, Cat.#80788, 1:1,000, Cell Signaling Technology), claudin-5 (ab131259, 1:2,000, Abcam), NANOG (sc-134218, 1:1,0000, Aanta Cruz), and GAPDH (60004-1-Ig, 1:1,000, Proteintech). The membranes were then washed three times with TBST for 10 min each and incubated with a secondary antibody (goat anti-mouse IgG-HRP antibody, ab205719, 1:20,000, Abcam; goat anti-rabbit IgG-HRP antibody, ab6721, 1:20,000, Abcam). Again after three washes with TBST, membranes were subjected to ECL chemiluminescence reagent (Thermo). Images were obtained using a ChemiDoc Gel Imaging system (Thermo), and the optical density was quantified with the Image J (v1.8.0) analytical software. Experiments were performed in triplicate.
10
Immunofluorescence Staining of Brain Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The prepared brain sections or cells were permeabilized with Triton X-100 and blocked using goat serum. Then the sections or cells were incubated with primary antibodies including anti-occludin (1:100, Abcam, Cambridge,United Kingdom), anti–p-NF-κB (1:100, Abcam, United Kingdom), anti–Iba-1 (1:200, Abcam, Cambridge,United Kingdom), anti–ZO-1 (1:100, ab216880, Abcam, United Kingdom), anti-PPARγ (1:100, ab272718, Abcam, United Kingdom), anti-iNOS (1:200, ab178945, Abcam,United Kingdom), anti-Arg1 (1:200, ab233548, Abcam, United Kingdom), and anti–claudin-5 (1:150, ab131259, Abcam, United Kingdom) overnight at 4°C. On the next morning, the sections or cells were incubated with goat anti-mouse or goat anti-rabbit secondary antibodies conjugated to Alexa 488 or Alexa 647 (1:200; Abcam, Cambridge, United Kingdom) for 1 h at 37°C. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Boyetime, Wuhan, China). After mounting, the immunofluorescent or immunohistochemical signals were observed under an Olympus microscope (Olympus, Tokyo, Japan). The positive cells were counted using Image J, with the researcher being blinded to the treatment conditions.
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!