Total proteins were extracted from cells and tissues with RIPA lysis buffer (Beyotime, P0013B) supplemented with phosphatase inhibitor tablets (Roche, 04906837001) and protease inhibitor tablet (Roche, 05892970001). The Enhanced BCA Protein Assay Kit (Beyotime, P0012) was used to assess the protein concentration, which was then adjusted to an equal concentration level in accordance with the manufacturer's instruction. The same amount of sample protein lysates was used in the standard western blot procedure to compare the expression of target proteins in different conditions. The following were the primary antibodies used in this experiment: anti-GAPDH (GNI, GNI4310-GH, 1:5000), anti-β-actin (Sigma, A5441), anti-Fibronectin (abcam, ab2413, 1:1000), anti-Periostin (R&D Systems, AF2955, 1:1000), anti-VEGFD (Proteintech, 26915-1-AP, 1:50), anti-Midkine (Proteintech, 11009-1-AP, 1:50), anti-SEMA3C (R&D Systems,MAB1728, 1:50), anti-VEGFR3 (R&D Systems, AF349, 1:50), anti-Nucleolin (Santa cruz, sc-8031, 1:50), anti-NRP2 (Neuropilin-2, CST, D39A5, 1:50), Vimentin (abcam, ab8978, 1:100), LYVE1 (abcam, ab281587, 1:100). The strips were then incubated with secondary antibody. As internal references, anti-GAPDH and anti-β-actin were used to calculate relative abundance of proteins.
Ab281587
Manufactured by Abcam
Sourced in United Kingdom
Ab281587 is a laboratory equipment product offered by Abcam. It is a device designed for specific laboratory applications. The core function of this product is to perform certain tasks required in a research or analytical setting. Further details about the intended use or specific capabilities of this product are not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Anti fibronectin, by Abcam (1 mentions)
Mab1728, by R&D Systems (1 mentions)
Anti periostin, by R&D Systems (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Protease inhibitor tablet, by Roche (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Phosphatase inhibitor tablet, by Roche (1 mentions)
Ab8978, by Abcam (1 mentions)
A2547, by Merck Group (1 mentions)
Dab substrate kit, by OriGene (1 mentions)
Ab49873, by Abcam (1 mentions)
3 protocols using ab281587
1
Protein Extraction and Western Blotting
2
Immunofluorescent Staining of Mouse Liver Tissue
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The frozen mouse liver tissue embedded in O.C.T. (#4583, SAKURA, Japan) was cut into 10 μm thick sections. The sections were fixed with acetone/methanol (4:1) for 10 min at − 20 °C, washed with TBS twice and blocked with 4% donkey serum for 1 h at room temperature (RT). Sections were then incubated with the indicated primary antibodies, including rabbit anti-collagen I (1:100; #PAB13488, Abnova, Taiwan, China), rabbit anti-GAS6 (1:200; #13795-1-AP, Proteintech, China), rabbit anti-integrin subunit beta 1 (ITGB1) (1:200; #26918-1-AP, Proteintech, China), rat anti-CD68 (1:100; #14-0681-82, eBioscience, USA), rabbit anti-desmin (DES) (1:200; #16520-1-AP, Proteintech, China) and rabbit anti-lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) (1:100; #ab281587, Abcam, UK) at RT for 1 h. After being washed with TBST thrice, the sections were then incubated with corresponding Donkey anti-Rat/Rabbit Alexa Fluor 488/594-conjugated secondary antibodies (1:1000; #A-21206-21209, Thermo, USA) for 1 h at RT. Finally, the sections were counterstained and mounted with DAPI, and observed with fluorescence microscopy.
3
Immunohistochemical Analysis of Liver Vasculature
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Mouse liver tissues were fixed in 10% neutral buffered formalin, dehydrated, and embedded in paraffin, as we previously reported.[22 , 23 ] Sections of 3.5‐µm thickness were incubated in citrate buffer (pH 6.0, BOSTER AR0024) for 15 min at 120ºC, and the endogenous peroxidase was blocked by 3% H2O2 for 10 min. The slides were incubated with 10% bovine serum albumin in PBS for 30 min at 37ºC to block the nonspecific binding sites, followed by incubation with appropriate primary antibodies for overnight at 4ºC, and then with horseradish peroxidase anti‐rabbit IgG or anti‐mouse IgG antibodies for 30 min. Color was then developed by incubation with DAB Substrate kit (ORIGENE ZLI‐9019). After washing in PBS, tissue sections were counterstained with hematoxylin and viewed under a microscope. Major primary antibodies used in the study include anti‐CD31 antibody (1:100; 77699s, CST), anti‐GS antibody (1:2000; ab49873, Abcam),[24 , 25 , 26 , 27 ] anti–lymphatic endothelial receptor‐1 (LYVE1) antibody (1:4000; ab281587, Abcam), and anti–α‐smooth muscle actin (α‐SMA) antibody (1:800; A2547, Sigma).
CD31+ portal vessels (excluding diameters ≤30 µm) and CD31+ central veins were manually counted based on staining of CD31 and GS. Six to eight randomly chosen fields from each section were analyzed.
CD31+ portal vessels (excluding diameters ≤30 µm) and CD31+ central veins were manually counted based on staining of CD31 and GS. Six to eight randomly chosen fields from each section were analyzed.
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