Five micrometer‐thick human liver sections (paraffin embedded) at different age stages were deparaffinized and rehydrated in graded alcohol. For antigen retrieval, liver sections were incubated in citric acid monohydrate buffer (Dako), then microwaved for 10 minutes at 720 W. Nonspecific staining was prevented by incubating the slices in 10% bovine serum albumin for 45 minutes at room temperature. Slices were incubated with anti‐NTCP at 1:100 (HP A042727; Sigma) and anti‐MRP2 at 1:50 (ALX‐801‐016‐C250; Enzo) primary antibodies overnight at 4°C. After the washing step, slices were incubated for 1 hour with donkey anti‐rabbit at 1:500 (A10042; Invitrogen) and then for one hour with donkey anti‐mouse at 1:500 (A31571; Invitrogen) secondary antibodies at room temperature. Nuclei were stained for 5 minutes with 4',6‐diamidino‐2‐phenylindole (Life Technologies). Slides were mounted in Fluoromount medium (Dako). Fluorescence was assessed using an Imager A1 fluorescence microscope (Carl Zeiss), and digital images were acquired using Axiovision software.
Hpa042727
Manufactured by Merck Group
Sourced in United States
HPA042727 is a laboratory equipment product from Merck Group. It is designed for scientific research and analysis purposes. The core function of this product is to provide a controlled environment for various experimental procedures. No further details about the intended use or specific applications of this product are available.
Automatically generated - may contain errors
Lab products found in correlation
A31571, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
A10042, by Thermo Fisher Scientific (1 mentions)
Ab3623, by Merck Group (1 mentions)
Hoechst, by Merck Group (1 mentions)
Axio imager z1 fluorescence microscope, by Zeiss (1 mentions)
Ab41898, by Abcam (1 mentions)
A0001, by Agilent Technologies (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Fluorescently labeled secondary antibody, by LI COR (1 mentions)
A1978, by Merck Group (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
Bs 1557g, by Bioss Antibodies (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Anti hnf4α, by Cell Signaling Technology (1 mentions)
Anti β actin a3854, by Merck Group (1 mentions)
5 protocols using hpa042727
1
Immunofluorescence Imaging of Liver Proteins
2
Immunofluorescence Analysis of Hepatocyte Markers
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Cells were fixed with 4% paraformaldehyde 4d pi and washed with PBS, permeabilized with 0.2% Triton-X in PBS (0.2% PBST) and blocked with 5% donkey serum in 0.2% PBST. The cells were stained overnight at 4°C with anti-HNF4α (Ab41898, Abcam), anti-Flavivirus Group Antigen (clone D1-4G2-4-1, Millipore), anti-ZIKV NS3 (kindly provided by Andres Merits, Institute of Technology, University of Tartu, Estonia), anti-albumin (ALB; A0001, Dako), anti-α-fetoprotein (AFP; A008, Dako), anti-sodium taurocholate cotransporting peptide (NTCP; HPA042727, Sigma-Aldrich) and anti-Caspase 3 (active, cleaved form) (AB3623, Millipore) antibodies. Afterwards, the cells were incubated for 30 min at room temperature with the appropriate secondary antibodies and Hoechst (Sigma-Aldrich). Images were taken using the AxioimagerZ.1 fluorescence microscope (Carl Zeiss Inc.). Appropriate isotype control antibodies were used in all immunofluorescence staining experiments.
3
SDS-PAGE and Western Blot Analysis of Viral and Host Proteins
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Cell lysates were applied to SDS-PAGE on 12% or 15% SDS gels. For NTCP-specific WBs, cell lysates were deglycosylated before SDS-PAGE with PNGAse F (New England Biolabs) according to the manufacturer’s protocol. Proteins were transferred onto nitrocellulose membranes by semidry transfer and incubated with primary antibodies (HDAg: serum of a chronically infected patient [VUDA, 1:3000 dilution]; HBsAg: human–anti-HBsAg [Humabs Biomed, kind gift from Davide Corti, 1:3000 dilution]; NTCP: rabbit–anti-NTCP [Sigma, HPA042727, 1:1000 dilution]; actin: mouse–anti-actin [Sigma, A1978, 1:5000 dilution]) diluted in 5% skim milk/TBST overnight at 4 °C. Membranes were washed with TBST and incubated with fluorescently labeled secondary antibodies (LI-COR Biosciences) for 1 h at RT, washed again, and imaged on a LI-COR Odyssey imaging system.
4
Immunostaining of Humanized Liver Chimeric Mouse
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Cryosections (6 μm) from humanized -liver chimeric mice were immunostained using an anti-human NTCP antibody (HPA042727, Sigma-Aldrich) and an anti-human cytokeratin (CK) 18 antibody (NB110-56910, Novus Biologicals, Littleton, CO). Formalin-fixed and paraffin-embedded liver sections (3 μm) were immunostained using an anti-HBs antibody (bs-1557 G, Bioss, Woburn, MA) and an anti-human albumin antibody (A80-129 A, Bethyl Laboratories Inc.).
5
Western Blot Analysis of Liver Proteins
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Cells were lysed in SDS buffer and incubated at 100 °C for 5 min. Proteins were separated in 10% SDS-PAGE and electro-transferred onto nitrocellulose membranes (Millipore, Billerica, USA). The membranes were blocked with 5% nonfat milk and incubated with specified primary antibodies [anti-β-actin (A3854, Sigma-Aldrich), anti-HNF4α (hepatocyte nuclear factor 4α) (C11F12, Cell Signaling Technology, Beverly, USA) and anti-SLC10A1 (solute carrier family 10 member 1/Na+/taurocholate cotransporting polypeptide/NTCP) (HPA042727, Sigma-Aldrich), anti-ALPL (alkaline phosphatase, liver/bone/kidney) (11187-1-AP, Proteintech, Rosemont, IL, USA), anti-GSTM1 (glutathione S-transferase mu 1) (12412-1-AP, Proteintech), anti-KYNU (kynureninase/l -kynurenine hydrolase) (11796-1-AP, Proteintech), anti-HKDC1 (hexokinase domain containing 1) (25874-1-AP, Proteintech)] overnight at 4 °C, followed by incubation with the corresponding secondary antibody. Signal detection was performed using ECL Blotting Substrate (Millipore). Each assay was repeated at least three times.
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