Total protein was extracted from cells using 200 µL 1 × SDS lysis buffer (P0013G; Beyotime Biotechnology Co., Shanghai, China) containing protease inhibitor. The concentration was determined using a BCA protein assay kit (BCA1-1KT; Sigma). Next, 30 µg protein was separated with 5% SDS-PAGE and transferred onto membranes. The membrane was blocked with 5% skimmed milk-TBST for 1 hour and probed overnight at 4°C with primary antibodies to HNF4A (ab92378; Abcam Inc., Cambridge, UK), CACNA1A (ab181371; Abcam), VEGFA (ab46154; Abcam), and β-actin (mAbcam 8226, 1: 5000; Abbkine, Redlands, CA, USA). After washing, the membrane was re-probed with the HRP-conjugated secondary antibody goat anti-rabbit (A0208; Beyotime) at 37°C for 45 minutes. Afterward, the membrane was visualized using an ECL reagent (ECL808-25; Biomiga), and the band intensities were analyzed using Gel-Pro Analyzer version 4.0 software (Media Cybernetics, Silver Springs, MD, USA). The ratio of the gray value of the target band to the internal reference β-actin was representative of the relative protein expression.
Ab92378
Manufactured by Abcam
Sourced in United Kingdom, Spain
Ab92378 is a lab equipment product from Abcam. It is designed for specific laboratory applications, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab182560, by Abcam (2 mentions)
Ab181371, by Abcam (1 mentions)
Gel pro analyzer version 4, by Media Cybernetics (1 mentions)
Ab46154, by Abcam (1 mentions)
Bca1 1kt, by Merck Group (1 mentions)
A0208, by Beyotime (1 mentions)
Ecl808 25, by Biomiga (1 mentions)
Ab32117, by Abcam (1 mentions)
Protease inhibitor, by Boster Bio (1 mentions)
Automatic imager, by Bio-Rad (1 mentions)
Ab6721, by Abcam (1 mentions)
Radio immunoprecipitation assay cell lysis buffer, by Boster Bio (1 mentions)
Bicinchoninic acid protein quantitative kit, by Boster Bio (1 mentions)
Ab8245, by Abcam (1 mentions)
Ab52625, by Abcam (1 mentions)
Ab7504, by Abcam (1 mentions)
Ab9021, by Abcam (1 mentions)
Leica tcs sp8 microscope, by Leica Biosystems (1 mentions)
A1 inverted confocal microscope, by Nikon (1 mentions)
Ti spinning disk confocal microscope, by Nikon (1 mentions)
5 protocols using ab92378
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Key Regulators
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The cultured cells were collected and lysed with enhanced Radio‐Immunoprecipitation assay cell lysis buffer containing protease inhibitor (Boster Biological Technology Co., Ltd.). Then, the bicinchoninic acid protein quantitative kit (Boster Biological Technology Co., Ltd.) was adopted to estimate the protein concentration. The protein was transferred to a polyvinylidene fluoride membrane after 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis. After 2‐h sealing with 5% bovine serum albumin at room temperature, the membrane was probed with primary rabbit anti‐human antibodies (Abcam) to HDAC2 (ab32117, 1:2000), HNF4A (ab92378, 1:1000), ARID1A (ab182560, 1:1000), and GAPDH (ab8245, 1:1000) overnight at 4℃. Horseradish peroxidase‐labelled goat anti‐rabbit immunoglobulin G (IgG) (ab6721, 1:5000, Abcam) was added into the membrane. After 1‐h incubation at room temperature, the membrane was developed with electrogenerated chemiluminescence working solution (EMD Millipore) and exposed using a Bio‐Rad automatic imager. ImageJ analysis software was adopted to quantify the grey value of each band in the Western blot image with GAPDH as a normalizer.
3
Immunofluorescence Staining of Liver Organoids
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To split f-IHOs, Matrigel® was digested with Trypsin-EDTA for 15 min at 37 °C. Cell suspension was centrifuged at 300g for 4 min, washed once with William’s E medium and resuspended in Matrigel® domes as described above. Organoids were typically passaged at a 1:4 ratio. For IF staining of organoids, Matrigel® was dissolved in cell recovery solution (11543560, ThermoFischer scientific) for 30 min at 4 °C and fixed for 30 min in 4% w/v PFA. Blocking was performed for 1 h in 3% donkey serum, 0.3% Triton and 0.1% DMSO. Primary antibodies were used 4 °C overnight at the indicated dilutions: CDH6 (AF2715, sheep, 1:50; RnD systems), EPCAM (ab7504, mouse, 1:100; abcam), KRT7 (ab9021, mouse, 1:100; abcam), HNF4A (ab92378, rabbit, 1:100; abcam), CK19 (ab52625, rabbit, 1:200; abcam), albumin (A80-129A, goat, 1:100; Bethyl), TROP2 (10428-MM02, mouse, 1:100; Stratech). DAPI was used at 1:1000 dilution as a counterstain. Alexa Fluor-conjugated secondary antibodies (all 1:500, Life Technologies). Images were acquired with a Nikon A1 inverted confocal microscope, a Nikon Ti spinning disk confocal microscope (Nikon Instruments Inc.) and a Leica TCS SP8 microscope (Leica Biosystems).
4
Immunohistochemical Analysis of Protein Expression
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The tissue specimens were embedded in paraffin, sectioned, dewaxed and hydrated. The sections were washed in 3% methanol H2O2 for 20 min, followed by antigen retrieval with citrate buffer in pressure cooker (2 min at 100℃ and 5 min at room temperature). Normal goat serum blocking solution (C‐0005, Shanghai Haoran Biotechnology Co., Ltd.) was dripped to the tissue sections. Then, the sections were placed at room temperature for 20 min. The liquid on the sections was dried. Primary rabbit anti‐human antibodies to HDAC2 (12922‐3‐AP, 1:200, Proteintech), HNF4A (ab92378, 1:500, Abcam), Ki‐67 (ab16667, 1:200, Abcam), and ARID1A (ab182560, 1:1000, Abcam) were added to the sections for overnight culture at 4℃, and the secondary antibody (ab6785, 1:1000, Abcam) was added. After incubation, protein working solution (0343‐10000U, Imun Biotechnology Co., Ltd.) was added into the sections and placed at 37℃ for 20 min. The sections were developed with diaminobenzidine (DAB; ST033, Whiga Biosmart Co., Ltd.), counterstained with haematoxylin (PT001, Shanghai Bogoo Biological Technology Co., Ltd.) for 1 min and blued with 1% ammonia water. The sections were observed and photographed under a microscope after sealing. Five visual fields were randomly selected from each section to observe and analyse the statistics.
5
Quantification of HNF4α expression in HepG2 cells
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Transfected HepG2 cells were washed twice with phosphate-buffered saline (PBS). They were then lysed in RIPA buffer [150 mMNaCl, 1 mMethylenediaminetetraacetic acid, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 20 mM 3-(N-morpholino) propanesulfonic acid (MOPS), 1 mMphenylmethylsulfonyl fluoride, pH 7.0] on ice for 20 min. The lysates were centrifuged at 12,000×g for 5 min at 4°C and the supernatants were collected for analysis of the protein concentration using a Bicinchoninic Acid (BCA) Protein Assay (Sigma-Aldrich, Madrid, Spain). These lysates (each 20 μg) were blotted and immunostained with different monoclonal antibodies: anti-HNF4α (ab92378; Abcam, Madrid, Spain) and anti-GAPDH (ab128915; Abcam, Madrid, Spain). HNF4α and GAPDH were immunodetected with the appropriate secondary antibody labeled with peroxidase (GE Healthcare, Barcelona, Spain). Western blotting detection, their corresponding densitometric analysis, and the expression of data were performed in a manner similar to that previously described [33 (link)].
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