After determining the protein concentration by Quick Start Bradford Protein Assay Kit (500-0202, Bio-Rad, Hercules, CA, USA), each vial of selected aqueous humor sample was supplemented with 5x SDS-PAGE loading buffer and denatured under 100°C for 5 min. The samples were separated by 15% gradient acrylamide sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel. Protein bands were transferred onto a PVDF blotting membrane (Millipore, Bedford, MA, USA) and subjected to immunolabeling using primary mouse anti-human antibodies for TGF-β2 (1 : 1000 dilution, ab36495, Abcam, Cambridge, MA, USA) and GAPDH (1 : 1000 dilution, ab8245, Abcam, Cambridge, MA, USA). The membranes were incubated with rabbit anti-mouse IgG-horseradish-peroxidase- (HRP-) conjugated secondary antibody (1 : 1000 dilution, ab6721, Abcam, Cambridge, MA, USA) for 30 min at room temperature. Immunoblotted bands were revealed by enhanced chemiluminescence reagent (Pierce ECL Western Blotting Substrate, Thermo Fisher Scientific, Waltham, MA, USA). The intensity of bands was measured using ImageJ 1.45s (USA).
Ab36495
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab36495 is a recombinant protein product. It is designed for use in laboratory research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab6721, by Abcam (3 mentions)
Pvdf blotting membrane, by Merck Group (1 mentions)
Quick start bradford protein assay kit, by Bio-Rad (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Ab8245, by Abcam (1 mentions)
Ab10805, by Abcam (1 mentions)
Ab92742, by Abcam (1 mentions)
Dmrb fluorescence microscope, by Leica (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Chemidoc imager, by Bio-Rad (1 mentions)
Immobilon p pvdf transfer membrane, by Merck Group (1 mentions)
M per mammalian extraction reagent, by Thermo Fisher Scientific (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (1 mentions)
Clarity max western ecl substrate, by Bio-Rad (1 mentions)
Ab6326, by Abcam (1 mentions)
Dfc360 fx camera, by Leica (1 mentions)
Dmi6000b inverted microscope, by Leica (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Cyanine3 (cy3), by Dianova (1 mentions)
12 protocols using ab36495
1
Quantification of TGF-β2 in Aqueous Humor
2
Immunofluorescence Staining of Tissue Samples
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Immunofluorescence staining was performed on 6-µm-thick frozen or paraffin-embedded tissue sections, as previously described (31 (link)). The following antibodies were used: Mouse monoclonal antibodies against TGF-β2 (#ab36495), vimentin (#ab8059) and 5-methylcytosine (#ab10805) (all from Abcam), and rabbit monoclonal antibodies against E-cadherin (#3195s; Cell Signaling Technology, Inc.) and Ki-67 (#ab92742; Abcam). Primary antibodies were diluted 1:50 and secondary antibodies 1:400; the incubations were all performed at room temperature. Signals were detected with a Leica DMRB fluorescence microscope (Leica Microsystems Ltd., Milton Keynes, UK). Images of representative fields were captured using a SPOTTM FLEX 15.2 64-Mp shifting pixel digital color camera and analyzed with SPOT Basic/Advanced 4.6 software (both from Diagnostic Instruments, Sterling Heights, Michigan, USA).
3
Western Blot Analysis of Protein Expression
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Five days after transfection or transduction, GTM3 or primary HTM cells were used for protein extraction using the M-PER Mammalian Extraction Reagent (Thermo Fisher Scientific). Equal amount of protein was mixed with 6× Laemmli buffer, boiled, and electrophoresed on SDS-PAGE gel. Separated proteins were transferred onto the Immobilon-P PVDF transfer membrane (Millipore, Burlington, MA, USA), blocked with 10% dry milk, and probed with one of the primary antibodies: mouse anti-TGFβ2 (1:500, ab36495; Abcam, Cambridge, UK), mouse anti-dCas9 (1:200, A-9000-100; Epigentek, Farmingdale, NY, USA), or rabbit anti-GAPDH (1:1000, 5174S; Cell Signaling Technology, Danvers, MA, USA) for 2 hours at room temperature. After washing with TBST, the blot was incubated with the secondary horseradish peroxidase (HRP) linked anti-mouse or anti-rabbit antibody (1:2000 or 1:10,000; Cell Signaling Technology) at 4°C overnight. The protein-antibody complex was incubated with the Clarity Max Western ECL substrate (Bio-Rad) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). Chemiluminescent signal was detected using the ChemiDoc imager (Bio-Rad).
Densitometry was conducted using the “Gels” tool (under the “Analyze” tab) in the ImageJ software (https://imagej.nih.gov/ij/ ; National Institute of Health, Bethesda, MD, USA). Naive bands were set at “1.00” for comparisons.
Densitometry was conducted using the “Gels” tool (under the “Analyze” tab) in the ImageJ software (
4
Immunocytochemistry of Oligodendrocyte Differentiation
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Primary oligodendroglial cells were cultivated for two days under proliferative conditions or for 6 days in differentiation medium on cover slips before fixation in 4% paraformaldehyde for 10 min. In some experiments, BrdU was added at a final concentration of 4 mM to proliferation medium 2 h before fixation. Cells on cover slips underwent immunocytochemistry using the following primary antibodies: rat anti-Mbp monoclonal (Bio-Rad, #MCA409S, 1:750 dilution), guinea pig anti-Sox10 antiserum (home made, 1:5000 dilution) (29 (link)), rabbit anti-Id4 (Novus Biologicals NBP2-56322, 1:50 dilution), mouse anti-Tgfb2 (Abcam ab36495, 1:50 dilution) and mouse anti-Wnt7a (Santa Cruz Biotechnology, E-9: sc-365665, 1:50 dilution). Secondary antibodies were coupled to Cy3 (Dianova, 1:400 dilution), Cy5 (Dianova, 1:400 dilution) or Alexa Fluor 488 (Molecular Probes, 1:0 000 dilution) fluorescent dyes. Incorporated BrdU was visualized using rat anti-BrdU (Abcam ab6326, 1:200 dilution). Stainings were documented with a Leica DMI6000 B inverted microscope (Leica) equipped with a DFC 360FX camera (Leica).
5
Immunoblotting of Aqueous Humor Proteins
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Pooled aqueous humor from WT and Tsk mice (n = 10–12 mice/group; 5 independent experiments) were used for immunoblotting. For immunoblotting, 10-30 µl of sample was heated for 5 min at 95 °C with reduced 6× Laemmli SDS sample buffer (Bioland Scientific LLC) and run in SurePAGE, 4–12% Bis–Tris gel (GeneScript). Immunoblot samples were loaded in equal protein concentrations for SDS-PAGE based on Bradford assays. The proteins were transferred onto PVDF membranes (Bio-Rad) and stained with Ponceau S staining solution (Bio-Rad) to verify transfer efficiency. Membranes were washed in Tris-buffered saline plus 0.1% Tween 20 (TBST) and blocked using 5% nonfat dry milk in TBST for 1 h at RT then incubated with primary antibodies (fibrillin-1 (ab231094, Abcam) or TGFβ2 (ab36495, Abcam)) overnight at 4 °C. After washing 3 times, membranes were incubated for 1 h at RT with HRP-conjugated secondary antibody (ab6789 or ab6721, Abcam) in blocking solution. Washed membranes were then incubated with a SuperSignal substrate (Thermo Scientific) for 1 min at RT and signal detection and densitometry analysis were performed with BioRad ChemiDoc XRS system.
6
Immunohistochemistry and Immunofluorescence Analyses in Uterine Tissues
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Immunohistochemistry and immunofluorescence analyses were performed as previously described42 (link). Uterine sections from paraffin-embedded tissues were preincubated with 10% normal serum in phosphate-buffered saline (PBS) and incubated with anti-TGF-β2 (ab36495; Abcam, Cambridge, MA, USA), anti-E-cadherin (610181; BD Bioscience, San Jose, CA, USA), anti-F4/80 (nbp2-12506; Novus, Littleton, CO, USA), anti-CD68 (SC-5474; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-CD4 (SC-1140; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-vimentin (ab92547; Abcam, Cambridge, MA, USA and SC-6260; Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies in 10% normal serum in PBS. On the following day, sections were washed in PBS and incubated with a secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. Immunoreactivity was detected using the Vectastain Elite DAB Kit (Vector Laboratories). Images were captured with a confocal microscope (510 NLO confocal microscope; Carl Zeiss, Thornwood, NY, USA).
7
Western Blot Analysis of EMT Markers
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The cells were lysed in the RIPA lysis buffer and electrically transferred to the polyvinylidene fluoride (PVDF) membranes, which were then blocked with 5% skimmed milk for 1 hour at room temperature (RT) and washed with TBST three times (10 min each time). Afterward, the membranes were incubated with the primary Anti-FAST1 antibody (1:1000, ab189960, Abcam, USA), Anti-E-Cadherin antibody (1:1000, ab231303, Abcam, USA), Anti-Claudin 1 antibody (1:1000, ab211737, Abcam, USA), Anti-N Cadherin antibody (1:1000, ab76057, Abcam, USA), Anti-Snail antibody (1:1000, ab82846, Abcam, USA), Anti-TGF-β (phospho S165) (1:1000, ab183037, Abcam, USA), Anti-TGF-β antibody (1:1000, ab36495, Abcam, USA), Anti-Smad2 (phospho S255) antibody (1:1000, ab188334, Abcam, USA), Anti-Smad2 antibody (1:1000, ab40855, Abcam, USA) and Anti-GAPDH antibody (1:1000, ab181603, Abcam, USA) at 4 °C overnight. After being rinsed with TBST, the membranes were incubated with the horseradish peroxidase (HRP)-labeled Goat Anti-Rabbit IgG (1:3000, ab6721, Abcam, USA) for 1 hour at RT. Then, the membranes were washed with TBST three times (10 min each time). Finally, ECL Western blot special reagents (Invitrogen) were used for color development and imaging, and Image J was applied to analyze each protein’s gray intensity.
8
Immunochemical Analysis of Breast Cancer Tissues
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Breast cancer tissues were cut into serial 5 μm sections and transferred onto electrostatic slides for immunochemical analysis. These specimens were heated at 65 °C for 30 min, deparaffinized in xylene, and rehydrated in a graded ethanol series. Subsequently, endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min. Next, 5% bovine serum albumin/1× Tris-buffered saline and Tween-20 were used to reduce nonspecific background staining. IHC staining of FOXP3 (mouse, clone 236A/E7, ab20034; Abcam, Cambridge, UK), interleukin (IL)-33 (rabbit, ab207737; Abcam), and transforming growth factor beta 2 (TGFB2) (mouse, clone SD4, ab36495; Abcam) was performed according to the manufacturer’s instructions. The degree of positivity of the markers was graded as low, medium, or high by one pathologist by visual estimation, and by two surgeons according to the H score. H scores <100, 100–199, and ≥200 were graded as low, medium, and high, respectively.
9
Protein Expression Analysis in Tissues and Cells
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RIPA lysis buffer was used to extract proteins from tissues or cell lines and protein samples (50 μg) were resolved by SDS-PAGE and then probed with indicated antibodies. CYB561D2, FAS ligand (FASLG), TGF-β2 (TGFB2), CD70, PD-L1, PD-L2, CCL2, TDO2, total STAT3, p-STAT3 (Tyr705) antibodies were from Sigma-Aldrich (SAB2500281), abcam (ab15285), abcam (ab36495), abcam (ab77868), abcam (ab205921), abcam (ab187662), abcam (ab186421), abcam (ab123403), CST (4904) and CST (9131), respectively. STAT3 inhibitor C188-9 was from selleckchem (S8605) [16 (link)]. The band density was analyzed with Image J and the density of target protein is normalized by Tubulin band. There are four biological replicates for each WB experiment.
10
Investigating Protein Interactions in U251 Cells
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To investigate the interaction between Aβ and CAV2, TGFB2 or TGFBR1, U251 cells were inoculated with β-amyloid (1–42), HiLyte Fluor™ 488-labelled (ANASPEC, AS-60479-01). After 1 h, the cells were washed, fixed and incubated with CAV2 (Abcam, ab133484), TGFB2 (Abcam, ab36495) or TGFBR1 antibodies (Abcam, ab31013). The cells were washed, counterstained with DAPI and observed under an Olympus FV1000 confocal laser microscope. To study the interaction between NEAT1 and P300/CBP, U251 cells were incubated with the NEAT1 probe overnight at 37 °C and then the anti-P300 (Abcam, ab59240) or anti-CBP antibodies (Abcam, ab50702) for 1.5 h at room temperature. After the cells were washed and incubated with the secondary antibody, they were counterstained with DAPI and mounted for observation. Cell images were obtained with an Olympus FV1000 confocal microscope. To study the role of NEAT1 in the interaction between STAT3 and H3K27Ac, U251 cells were transfected with a NEAT1 or negative control siRNA for 36 h and then incubated with the anti-STAT3 Y705 (Abcam, ab76315) and anti-H3K27Ac antibodies (Abcam, ab4729) for 1.5 h at room temperature. After the cells were washed and incubated with the secondary antibody, they were counterstained with DAPI and mounted for observation. Cell images were obtained with an Olympus FV1000 confocal microscope.
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