Total protein from cells was extracted by lysis and resolved by 8–12% SDS-PAGE. Samples were then electro-transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Boston, MA, USA). PVDF membranes were blocked with 5% BSA or skimmed milk. Target proteins were detected with antibodies against GAPDH (Abcam, ab9485, diluted 1:2500), NOX1 (Abcam, ab78016, diluted 1:1000), NOX2 (Abcam, ab31092, diluted 1:1000), NOX3 (Abcam, ab82708, diluted 1:2000), NOX4 (Abcam, ab133303, diluted 1:2000), NOX5 (Abcam, ab198213, diluted 1:2000), DUOX1 (Abcam, ab178534, diluted 1:5000), DUOX2 (Abcam, ab170308, diluted 1:500), VEGFR-1 (Abcam, ab32152, diluted 1:2000), VEGFR-2 (Abcam, ab11939, diluted 1:1000), VEGFR-3 (Abcam, ab27278, diluted 1:1000), EGFR (Abcam, ab52894, diluted 1:5000), p-EGFR (Abcam, ab40815, diluted 1:2000), PDGFR-α (Abcam, ab203491, diluted 1:1000), PDGFR-β (Abcam, ab32570, diluted 1:10000) and C-Met (Abcam, ab51067, diluted 1:5000) overnight at 2–8 °C, followed by incubation with specific horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam, ab6721, diluted 1:10,000) for 2 h at room temperature. Bands were visualized by chemiluminescence with enhanced chemiluminescent detection reagents (Millipore, Boston, MA, USA).
Ab27278
Manufactured by Abcam
Sourced in United States, China, United Kingdom
Ab27278 is a primary antibody targeting the human CD45 protein. CD45 is a receptor-linked protein tyrosine phosphatase that is expressed on the surface of most hematopoietic cells.
Automatically generated - may contain errors
Lab products found in correlation
Ab6721, by Abcam (2 mentions)
Ab9485, by Abcam (2 mentions)
Ab32570, by Abcam (1 mentions)
Ab203491, by Abcam (1 mentions)
Ab40815, by Abcam (1 mentions)
Ab11939, by Abcam (1 mentions)
Ab52894, by Abcam (1 mentions)
Ab133303, by Abcam (1 mentions)
Enhanced chemiluminescent detection reagent, by Merck Group (1 mentions)
Ab78016, by Abcam (1 mentions)
Ab51067, by Abcam (1 mentions)
Ab31092, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab32152, by Abcam (1 mentions)
Ab25377, by Abcam (1 mentions)
Ab5103, by Abcam (1 mentions)
Cm1860 uv, by Leica camera (1 mentions)
Ab182651, by Abcam (1 mentions)
Pvdf membrane, by Roche (1 mentions)
A0453, by Beyotime (1 mentions)
10 protocols using ab27278
1
Western Blot Analysis of NADPH Oxidase Proteins
2
Imaging Lymphatic Drainage After IVH
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IF staining was performed as previously described. Briefly, 24 h after IVH, the mice were subjected to deep anesthesia and perfused with physiological saline and 4% paraformaldehyde. The meninges and dCLNs were isolated. Then, the meninges and dCLNs were immersed in 4% paraformaldehyde for 24 h and subsequently dehydrated in a 30% sucrose solution for 72 h. Lymph node sections (15 μm) were obtained using a cryostat (Leica, CM1860UV, Germany) and treated with 0.5% Triton for 30 min. Subsequently, the lymph node slices and meninges were blocked with 5% normal goat serum for 2 h and incubated overnight at 4 °C with the following primary antibodies: anti-LYVE-1 (rabbit, 1:200, 67538S, Cell Signaling Technology), anti-LYVE-1 (rat, 1:100, 140-443-82, Thermo Fisher), anti-CX3CR1 (rabbit, 1:100, 13885-1-AP, Proteintech), anti-CitH3 (rabbit, 1:200, ab5103, Abcam), anti-Ly6G (1:200, ab25377, Abcam), anti-VEGFR3 (rabbit, 1:100, ab27278, Abcam), and anti-fibrinogen (sheep, 1:200, PA185429, Thermo Fisher). Next, the samples were incubated with the appropriate secondary antibodies at room temperature for 2 h, followed by staining with DAPI for 5 min.
3
Protein Expression Analysis in Tumor Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted from tumor tissues and cells, and denatured and quantified. Protein samples were then separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Roche, Basel, Switzerland). After blocking the membranes with 5% skimmed milk for 2 h, the membranes were incubated with primary antibodies against VEGF-C (1:1000, CST2445, CST, Boston, MA, USA), VEGFR-3 (1:1000, ab27278, Abcam, USA), MMP-2 (1:1000, CST87809S, CST), MMP-7 (1:1000, 10374-2-AP, Proteintech), MMP-9 (1:1000, CST13667S, CST), PI3K (1:1000, 60225-1-1g, Proteintech), p-PI3K (1:1000, ab182651, Abcam), AKT (1:1000, 60203-2-1g, Proteintech), p-AKT (1:1000, 66444-1-1g, Proteintech), or GAPDH (1:5,000, 60004-1-lg, Proteintech) overnight at 4˚ C and subsequently with the appropriate HRP-conjugated secondary antibody followed by enhanced chemiluminescence detection.
4
Identifying Lymphatic Endothelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunostaining was performed as previously described (Wang et al., 2018 (link)). mLECs were identified by immunostaining with anti-LYVE-1 (NBP1-43411, Novus) and anti-VEGFR-3 (ab27278, Abcam) antibodies, as both of which are the selective markers for lymphatic endothelium. Briefly, cells were washed with PBS three times, fixed with 4% paraformaldehyde for 30 min, permeated with 0.1% Triton X-100 for 15 min, and then blocked with 5% bovine serum albumin. Cells or heart frozen sections were incubated with primary antibodies against LYVE-1 and VEGFR-3 (diluted 1:100) overnight at 4°C and then incubated with a red fluorescent secondary antibody (diluted 1:200; A0453, Beyotime) for 2 h at room temperature. The cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and are shown in blue. The images were analyzed using a Nikon microscope (Tokyo, Japan).
5
Evaluating BRAFV600E and VEGFR Expression in BHT-101 Tumors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ex vivo immunohistochemistry was performed to evaluate changes in morphology as well as BRAFV600E and VEGFR expression in BHT-101 tumors. Mice bearing BHT-101 tumors were treated with onalespib and/or sorafenib as previously described (n = 12). When tumors reached a size of 1000 mm3 or day 25 (endpoint of experiment), tumors were collected and fixed in 4% buffered formalin and transferred to 70% ethanol. Tumor tissues were paraffin embedded, sectioned, and deparaffinized. Immunohistochemical stainings were performed on FFPE sections using fully automated protocols (DAKO Autostainer Link48) and Envision FLEX, high pH detection kit from DAKO #K8000. The following antibodies were used: BRAF V600E (Sigma Aldrich, #SAB560047, rabbit monoclonal, clone RM8, diluted 1:2000) and VEGFR3 (abcam, #ab27278, polyclonal, diluted 1:400). Immunohistochemical sections were manually scored according to staining intensity (negative −, weak +, moderate ++, or strong +++).
6
Podoplanin and VEGFR-3 Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted using radioimmunoprecipitation assay buffer and the protein concentration was established using the Bradford Protein Assay Kit (Bio Basic). Protein samples were diluted in 4X Laemmli buffer, then heated at 95°C for 5 minutes. Proteins were separated by electrophoresis on a 12% SDS‐PAGE, then transferred on nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in Tris‐buffered saline (TBST, 0.1% Tween 20) for 1 hour at room temperature, then incubated with an anti‐podoplanin (OriGene, DP3512S), an anti‐vascular endothelial growth factor receptor (VEGFR)‐3 (Abcam, AB27278), or an anti‐beta‐actin (Abcam, AB8227) overnight at 4°C. The membranes were washed with TBST and incubated with horseradish peroxidase–conjugated secondary antibodies (Abcam, AB6721 and AB6721) for 1 hour at room temperature. Western Lightning Ultra chemiluminescence kit (PerkinElmer) was used for detection. Each sample was normalized with its respective beta‐actin expression.
7
Sirtuin 3 Overexpression Regulates Angiogenesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sirtuin 3 overexpressing lentivirus was purchased from GenePharma (GenePharma). Mini‐osmotic pump was purchased from Alzet (Durect). Ang II was purchased from Sigma (Sigma Aldrich). Mouse VEGFC ELISA kit was purchased from Meimian (Meimian). Lipofectamine 2000 was purchased from Invitrogen (Thermo Fisher). DMEM was purchased from Sciencell (Sciencell). Foetal bovine serum was purchased from Gibco (Thermo Fisher). EDU Apollo Imaging Kit was purchased from Ribobio (Ribobio). Antibodies against SIRT3, tubulin and GAPDH were purchased from Cell Signaling Technology (D22A3, 5335S and 5174S, CST). Antibody against ERK, p‐ERK and MYH6 was purchased from Proteintech (16443‐1‐AP, 80031‐1‐AP and 22281‐1‐AP, ProteinTech). Antibodies against LYVE1, VEGFR3 and α‐SMA were purchased from Abcam (ab19147, ab27278 and ab5694, Abcam). Mouse monoclonal antibody against SIRT3 was purchased from Santa Cruz (sc‐365175).
8
Immunohistochemical Analysis of Lymphatic Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three mice in each experimental group were randomly selected. After fixing the tumor tissues in 4% polymethylene formaldehyde for 24 h, they were cut into 4 μm sections. After the waxing was removed, the slides were treated accordingly using KIT-9710 (MXB Biotechnologies, Fuzhou, China). The slides were incubated with the primary antibodies against LYVE-1 (1:200, ab33682, Abcam), VEGF-C (1:100, CST2445, CST, Boston, MA, USA), or VEGFR-3 (1:100, ab27278, Abcam, Shanghai, China). After antibody incubation, slices were stained with DAB (DAB-0031, MXB Biotechnologies) and hematoxylin (G1140, Solaibao, Beijing, China). Finally, three fields were randomly selected under the optical microscope (400×), in which the brown-yellow particles were observed, then analyzed with Motic 6.0 image analysis system.
9
Quantitative Immunoblotting of Inflammatory Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To measure the levels of interleukin (IL)-6, IL-1β, tumor necrosis factor-α (TNF-α), VEGF-C, VEGFR3, and LYVE-1, equal amounts of the samples were loaded onto 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels. Then, the resolved proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and incubated for one hour, at room temperature, in blocking solution (5% nonfat dried milk dissolved in Tris-buffered saline with Tween 20 [TBST] buffer). Then, the filters were probed, overnight, at 4°C, in blocking solution containing primary antibodies, diluted 1 : 1,000, against the following: IL-6 (MB9296, Bioworld), IL-1β (BS6067, Bioworld), TNF-α (11948S, Cell Signaling Technology), VEGF-C (sc-374628, Santa Cruz Biotechnology), VEGFR3 (ab27278, Abcam), LYVE-1 (ab14917, Abcam), and β-actin (AP0060, Bioworld). Membranes were washed twice with TBST buffer and incubated with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology), for 1 hour, at room temperature, followed by washing three times. Signal detection was performed using an enhanced chemiluminescence substrate (Millipore, USA) and quantitated using Image J software.
10
Protein Expression Analysis in Decidual Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was used to detect the expression levels of cleaved caspase-3, Fas, FasL, and VEGF in decidual tissues of the mice. Decidual tissue homogenate was again prepared for western blotting. Protein extraction was performed using RIPA cell lysis reagent (BB-3209; BestBio Technology, Co., Ltd., Shanghai, China). The proteins were separated with SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane at a constant voltage of 80 V. After being sealed by blocking solution for 1 h, the membrane was incubated with anti-rabbit primary antibodies against cleaved caspase-3 (1: 500; ab49822; Abcam, Cambridge, UK), Fas (1: 1,000; ab15285; Abcam), FasL (1: 1,000; ab15285; Abcam), VEGF (1: 1,000; Ab27278; Abcam) and GAPDH (1: 2,500; ab9485; Abcam), and sealed for incubation with shaking overnight at 4°C. Then, the membrane was incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1: 2,000; ab205718; Abcam) with shaking at 37°C for 2 h. The PVDF membrane was color-developed and photographed. The relative expression of a target protein = the gray value of the target band/the gray value of the internal control band.
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!