Cells were lysed by radioimmunoprecipitation assay (RIPA) buffer (Pierce) on ice to obtain total protein. Samples were centrifuged at 14,000g for 15 min at 4 °C and then heated at 95 °C for 5 min in 1× SDS loading buffer. We separated the protein by using 8% or 10% SDS–polyacrylamide gels and transferred it to PVDF membranes by a wet transfer apparatus (Bio-Rad). The membranes were blotted with 5% milk or bovine serum albumin (BSA) for 1 h and then incubated with primary antibodies at 4 °C overnight (70 (link)). The following antibodies were used: rabbit anti-ALKBH1 (1:2000, ab128895, Abcam), rabbit anti-HIF-1α (1:1000, ER1802-41, Huabio, Boston), rabbit anti-GYS1 (1:1000, ET1611-59, Huabio), rabbit anti-VEGF (1:2000, ET1604-28, Huabio), rabbit anti-GLUT1 (1:2000, ET1601-10, Huabio), rabbit anti-LDHA (1:2000, ET1608-57, Huabio), rabbit anti-CEBPA (1:2000, ET1612-46, Huabio), rabbit anti-FABP4 (1:2000, ET1703-98, Huabio), mouse anti-α-tubulin (1:2,000, sc-32293; Santa Cruz Biotechnology). The complexes were incubated in horseradish peroxidase conjugated anti-rabbit or anti-mouse IgG secondary antibodies (Cell signaling Technology) and visualized with Immobilon reagents (Millipore).
Et1703 98
Manufactured by Huabio
Sourced in China
The ET1703-98 is a laboratory instrument designed for performing scientific analysis and measurements. It is a core piece of equipment used in various research and testing applications. The detailed specifications and intended use of this product are not available without further information.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon reagent, by Merck Group (1 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse igg secondary antibody, by Cell Signaling Technology (1 mentions)
Sc 32293, by Santa Cruz Biotechnology (1 mentions)
Wet transfer apparatus, by Bio-Rad (1 mentions)
Ab128895, by Abcam (1 mentions)
Ba1772, by Boster Bio (1 mentions)
Envision hrp kit, by Agilent Technologies (1 mentions)
Rt1212, by Huabio (1 mentions)
Ba0325, by Boster Bio (1 mentions)
Ab5694, by Abcam (1 mentions)
Ab955, by Abcam (1 mentions)
2 protocols using et1703 98
1
Protein Expression Analysis by Western Blot
2
Histological Analysis of Kidney Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Commercial kits (Sigma-Aldrich, United States) were used for Masson or HE staining of kidney tissue according to the manufacturer’s protocol. Masson staining and immunostaining intensity were scored by photograph, randomly selected under 200 magnified visual field with a light microscope. According to the degrees of tubule degeneration and necrosis, tubule atrophy, inflammatory cell infiltration, and fibrosis, 0, 1, 2, and 3 were scored. The collagen-blue area representing the lesion area after Masson staining was calculated. The staining area was measured by the average optical density of the immunostaining intensity score. The ratios of FABP4-positive area to total area in kidney in glomeruli at a magnification of ×400 and in the cortical tubulointerstitium except glomeruli and vascular lumen at a magnification of ×200 were calculated as FABP4 positive in glomerular and FABP4 positive in interstitial, respectively.
Immunohistochemical staining was performed on 4-μm kidney sections as previously described. After antigen retrieval, sections were incubated with the primary antibody against α-SMA (ab5694; Abcam, United States), Col-1 (BA0325; Bosterbio, United States), FN (BA1772; Bosterbio), FABP4 (ET1703-98, HuaBio, China), F4/80 (RT1212; HuaBio), or CD68 (ab955; Abcam), respectively, and then detected by the En Vision/HRP Kit (Dako, Carpinteria, CA, United States).
Immunohistochemical staining was performed on 4-μm kidney sections as previously described. After antigen retrieval, sections were incubated with the primary antibody against α-SMA (ab5694; Abcam, United States), Col-1 (BA0325; Bosterbio, United States), FN (BA1772; Bosterbio), FABP4 (ET1703-98, HuaBio, China), F4/80 (RT1212; HuaBio), or CD68 (ab955; Abcam), respectively, and then detected by the En Vision/HRP Kit (Dako, Carpinteria, CA, United States).
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