Primary HASMCs, primary HUVECs, SMC growth medium-2 (SmGM-2), and endothelial cell growth medium-2 (EGM-2) were supplied from Lonza (Basel, Switzerland). THP-1 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). RPMI 1640 and Dulbecco’s modified Eagle’s medium (DMEM) were ordered from Corning (New York, NY, USA), and human plasma fibronectin was purchased from Millipore (Burlington, MA, USA). Sixteen percentage formaldehyde and Hoechst 33342 were obtained from Thermo Fisher Scientific. Trichloro(1H,1H,2H,2H-perfluorooctyl)silane, erioglaucine, Triton X-100, and albumin from bovine serum (BSA) were bought from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-smooth muscle myosin heavy chain 11 antibody (ab133567, 1:25), mouse anti-CD31 antibody conjugated with Alexa Fluor 488 (ab215911, 1:100), mouse anti-intercellular adhesion molecule 1 (ICAM1) antibody (ab2213, 1:50), rabbit anti-von Willebrand factor antibody (ab6994, 1:100), goat anti-mouse IgG H&L Alexa Fluor 488 (ab150113, 1:200), and goat anti-rabbit IgG H&L Alexa Fluor 594 (ab150080, 1:200) were ordered from Abcam (Cambridge, UK). Recombinant human TNF-α was supplied from PeproTech (Rocky Hill, NJ, USA). A PDMS polymeric base and a curing agent were purchased from Dow Chemical Company (Midland, MI, USA).
Ab133567
Manufactured by Abcam
Sourced in United Kingdom
Ab133567 is a recombinant monoclonal antibody. It is designed for use in various immunoassays to detect the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Ab14106, by Abcam (3 mentions)
Ab5694, by Abcam (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Ab6994, by Abcam (1 mentions)
Human plasma fibronectin, by Merck Group (1 mentions)
Ab2213, by Abcam (1 mentions)
Ab150080, by Abcam (1 mentions)
Albumin from bovine serum bsa, by Merck Group (1 mentions)
Goat anti mouse igg h l alexa fluor 488, by Abcam (1 mentions)
Primary huvec, by Lonza (1 mentions)
Erioglaucine, by Merck Group (1 mentions)
Ab150113, by Abcam (1 mentions)
Recombinant human tnf α, by Thermo Fisher Scientific (1 mentions)
Thp 1 cells, by Korean Cell Line Bank (1 mentions)
Goat anti rabbit igg h l alexa fluor 594, by Abcam (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Corning (1 mentions)
Rpmi 1640, by Corning (1 mentions)
6 protocols using ab133567
1
Vascular Cell Coculture Protocol
2
Immunoblotting Analysis of Apoptosis and Fibrosis Markers
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Subsequently, using the RIPA buffer, proteins were extracted from cells for immunoblotting. 15–50 μg of total protein extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After blocked with 5% skimmed milk, membranes were then probed with anti-Bax (1:2,000 dilution; #ab3203; Abcam, Cambridge, UK), anti-Bcl-2 (1:1,000; #ab32124; Abcam), anti-cleaved-caspase3 (1:500; #ab32042; Abcam), anti-cleaved-caspase9 (1:1,000; #ab2324; Abcam), anti-α-SMA (1:1,000; #ab5694; Abcam), anti-SM22 (1:1,000; #ab14106; Abcam), anti-SM-MHC (1:2,000; #ab133567; Abcam), anti-vimentin (1:2,000; #ab92547; Abcam), anti-collagen I (1:1,000; #ab270993; Abcam), anti-SMAD3 (1:2,000; #ab40854; Abcam), and anti-β-actin (1:5,000; #ab8226; Abcam) at room temperature for 1.5 h. Then, membranes were incubated with the appropriate secondary antibody conjugated to HRP. Then, the BM chemiluminescence blotting system (Thermo Fisher Scientific, Waltham, MA, USA) was used to visualize protein bands, and ImageJ Software (NIH, Bethesda, MD, USA) was used to quantify protein bands.
3
Angiotensin II-Induced HASMC Phenotypic Changes
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After treatment with angiotensin II and indicated transfection, HASMCs were incubated for additional 48 hours, lysed on ice for 30 minutes using RIPA buffer supplemented with protease inhibitors (A32963, ThermoFisher Scientific), and centrifuged for 30 minutes at 4 °C. Cell lysates were harvested. After the determination of protein concentration, 40 μg protein was electrophoresed in 10% SDS‐PAGE and transferred to polyvinylidene difluoride membrane (10600069, GE Healthcare, Pittsburgh, PA). The membrane was then blocked and incubated with primary antibodies against human α‐SMA (ab5694, 1:1000; Abcam), SM22α (ab14106, 1:1000; Abcam), osteopontin (ab8448, 1:1000, Abcam), MYH11 (ab133567, 1:500, Abcam), TGFβR1 (3712, 1:1000, Cell Signaling Technology), and GAPDH (AC001, 1:5000; ABclonal, Wuhan, China) for 2 hours at room temperature. The membranes were then washed, probed with horseradish peroxidase‐conjugated secondary antibodies, and visualized with enhanced luminol‐based chemiluminescent substrate (W1001, Promega). The bands were quantified with National Institutes of Health ImageJ software (version 1.52s).
4
Western Blot Analysis of VSMC Markers
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VSMCs in each group were collected and lysed in 100 μL of RIPA lysis buffer (Beyotime, Shanghai, China). Then, the total protein was extracted and the protein concentration was determined by Bradford method. The equivalent amount of protein in each group was separated by SDS-PAGE and then transferred onto PVDF membrane (Millipore, Bedford, MA, USA). Then, the membrane was blocked in 5% skim milk at room temperature for 1 h before being incubated with the primary antibody overnight at 4°C. Next, secondary antibody was added and the membranes were incubated at room temperature for 1 h. Electrochemiluminescence substrate A and B solutions (Millipore, Bedford, MA, USA) and Amersham Imager 600 (GE Healthcare, Chicago, IL, USA) were used to visualize the protein bands. The ImageJ software (NIH, Bethesda, Maryland, USA) was utilized for quantifying the protein bands. The antibodies used in this study included anti-PIK3CG (1:1000, 140,307, Abcam, Cambridge, UK), anti-SM-22α (1:1000, ab14106, Abcam, Cambridge, UK), anti-α-SMA (1:1000, ab184705, Abcam, Cambridge, UK), anti-smooth muscle myosin heavy chain 11 (SMMHC) (1:1000, ab133567, Abcam, Cambridge, UK), anti-Calponin (1:1000, ab227661, Abcam, Cambridge, UK), anti-GAPDH (1:3000, 60,004-1-Ig, Proteintech, Wuhan, China), and the secondary antibody (1:2000, SA00001-2, Proteintech, Wuhan, China).
5
Histological and Immunohistochemical Analysis of Vascular Grafts
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The vascular grafts or regenerated vessels explanted from monkeys and pigs were processed for histological and immunohistochemical analyses as described previously (21). The following antibodies were used: rabbit anti-Oct4 polyclonal antibody (Abcam, US, ab19857), mouse anti-CD31 monoclonal antibody (Maixin, China, MAB-0031), goat anti-CD31 (R&D, US, AF3628), rabbit anti-α-SMA polyclonal antibody (Abcam, US, ab5694), rabbit anti-calponin monoclonal antibody (Abcam, US, ab46794), rabbit anti-SMM-HC (Myh11) monoclonal antibody (Abcam, US, ab133567), rabbit anti-collagen I polyclonal antibody (Abcam, US, ab34710), rabbit anti-PGP9.5 monoclonal antibody (Abcam, US, ab108986), donkey anti-goat IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 488 (Thermo Fisher, US, A11055), goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 568 (Thermo Fisher, US, A11011), and SignalStain Boost IHC Detection Reagent (HRP, Rabbit/Mouse) (Cell Signaling Tech., Tech., US, 8114/8125). All the tissues prepared for staining were harvested from the middle part of the implants.
6
Immunofluorescence Analysis of Lung Tissue
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Rat lung tissues and were fixed in 4% paraformaldehyde, and lung tissue paraffin sections were prepared according to the SOP. After dewaxed and hydrated, the sections were incubated in QuickBlock™ blocking buffer (Beyotime, Shanghai, China) for 30 min at room temperature. Similarly, BMSCs after intervention were prepared as cell slides. Sections of lung tissue were subjected to anti- Sexdetermining Region Y (SRY; Abcam ab140309; 1/100) and anti-CD34 antibody (Abcam, ab4648; 1/100), the cell slides were subjected to anti-SRY, α-actin (abclonal, A7248; 1/100), calponin3 (Abcam, ab204365; 1/100), SM-MHC (Abcam, ab133567; 1/100), CD34 (Abcam, ab81289; 1/100), CD105 (Abcam, ab2529; 1/100), overnight incubation at 4 °C, and washed three times with phosphate buffered saline (PBS) after incubation. Secondary antibody incubation was performed with HRP-labeled goat anti-rabbit IgG (Servicebio, GB23303, 1/100) and Cy3-labeled goat anti-rabbit IgG (Servicebio, GB21303, 1/100), Nuclei were counterstained with DAPI (Sigma-Aldrich, St Louis, MO). After all staining was completed, the lung tissue staining was observed using a BX53 fluorescence microscope (Olympus, Tokyo, Japan) at 400× magnification.
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