Cells were fixed with 4% PFA and incubated with primary antibodies against albumin (Bethyl A90-234A), α-SMA (Sigma A2547), Cyp1a2 (Abcam ab22717) or γH2A.X (Abcam ab26350) followed by the appropriate secondary antibodies conjugated to Alexa Fluor 555 (Invitrogen A31570) or Alexa Fluor 488 (Invitrogen A11078). Images were taken with an Olympus IX51 inverted fluorescent microscope. For F-actin staining, cells were fixed with 3.7% PFA and incubated with fluorescent phallotoxins (Gibco A34055). Nuclei were stained with Hoechst (Sigma 33342).
Ab22717
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab22717 is a lab equipment product offered by Abcam. It is a device designed for use in scientific research applications. The core function of this product is to facilitate a specific experimental or analytical process, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab26350, by Abcam (1 mentions)
Hoechst, by Merck Group (1 mentions)
Ix51 inverted fluorescent microscope, by Olympus (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Ab81087, by Abcam (1 mentions)
Ab4236, by Abcam (1 mentions)
Ab137015, by Abcam (1 mentions)
Ix71 inverted fluorescent microscope, by Olympus (1 mentions)
Ab19194, by Abcam (1 mentions)
Ab76055, by Abcam (1 mentions)
Ab49355, by Abcam (1 mentions)
Ab15580, by Abcam (1 mentions)
Sc 8987, by Santa Cruz Biotechnology (1 mentions)
Ab8227, by Abcam (1 mentions)
Ab88443, by Abcam (1 mentions)
Goat anti mouse igg hrp antibody, by Santa Cruz Biotechnology (1 mentions)
2 amino 3 methylimidazo 4 5 f quinoline iq, by LGC (1 mentions)
Ab52642, by Abcam (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
7 protocols using ab22717
1
Immunofluorescence Staining of Liver Cells
2
Immunofluorescence Staining of Cells and Tissue Sections
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Cells were fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature, after being washed with PBS three times, the cells were blocked and permeated with PBS containing 5% BSA and 0.3% Triton for 1 hr at room temperature. Then cells were incubated with the relevant primary antibody at 4°C overnight. After a thorough washing, cells were incubated with the appropriate fluorescence-conjugated secondary antibody for 1 hr at room temperature. Nuclei were stained with Hoechst 33,342 (10 μg/mL).
For staining of frozen tissue sections, fresh liver specimens were embedded in cryo-embedding medium and stored in frozen blocks at −80°C prior to sectioning. Sections of 10 μm thickness were fixed with 4% PFA for 30 min, and then blocked with 5% BSA. The sections were then incubated with the correct primary antibodies and secondary antibodies similar to cell staining. Nuclei were stained with Hoechst 33,342 (10 μg/mL). Images were captured with an Olympus IX71 inverted fluorescent microscope. Antibodies used in this study are as follows: anti-E-cadherin (CST, 24E10), anti-albumin (Bethyl Laboratories, A90-234A), anti-ZO-1 (Gibco, 40–2200), anti-Cyp1a2 (Abcam, ab22717), anti-Cyp2c9 (Abcam, ab4236), anti-Cyp2c19 (Abcam, ab137015), and anti-Fah (Abcam, ab81087).
For staining of frozen tissue sections, fresh liver specimens were embedded in cryo-embedding medium and stored in frozen blocks at −80°C prior to sectioning. Sections of 10 μm thickness were fixed with 4% PFA for 30 min, and then blocked with 5% BSA. The sections were then incubated with the correct primary antibodies and secondary antibodies similar to cell staining. Nuclei were stained with Hoechst 33,342 (10 μg/mL). Images were captured with an Olympus IX71 inverted fluorescent microscope. Antibodies used in this study are as follows: anti-E-cadherin (CST, 24E10), anti-albumin (Bethyl Laboratories, A90-234A), anti-ZO-1 (Gibco, 40–2200), anti-Cyp1a2 (Abcam, ab22717), anti-Cyp2c9 (Abcam, ab4236), anti-Cyp2c19 (Abcam, ab137015), and anti-Fah (Abcam, ab81087).
3
Antibody Characterization for Hepatocyte Study
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The following antibodies were used: rabbit polyclonal anti-HNF4α (sc-8987, Santa Cruz, California, CA, USA), goat polyclonal anti-ALB (ab19194, Abcam, Cambridge, MA, USA), rabbit polyclonal anti-ASGR1 antibody (ab49355, Abcam Cambridge, MA, USA), mouse monoclonal anti-CYP1a2 (ab22717, Abcam Cambridge, MA, USA), rabbit polyclonal anti-β-actin (ab8227, Abcam Cambridge, MA, USA), mouse monoclonal anti-E-cad (ab76055, Abcam Cambridge, MA, USA), and rabbit polyclonal anti-Ki-67 (ab15580, Abcam Cambridge, MA, USA). The detailed information of primary antibodies used for immunoblotting assay and immunohistochemistry are summarized in Table 1 .
4
Yeast and Bacterial Strain Culture
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Standard media were used for the culture of yeast and bacterial strains (Burke et al. 2000 ). The bacterial DH1 strains containing the vectors pCS316 and pCYP1A2_NAT2 were cultured in LB-Amp (Luria Broth media with 100 µg/mL ampicillin). Media used for the culture of yeast cells included YPD (yeast extract, peptone, dextrose), SC (synthetic complete, dextrose), and SC-URA (SC lacking uracil). A mouse monoclonal antibody to CYP1A2 (ab22717) and a mouse polyclonal antibody to NAT2 (ab88443) were purchased from Abcam. Purified recombinant NAT2 protein was purchased from OriGene (TP761755). A goat antimouse IgG-HRP antibody was purchased from Santa Cruz Biotechnology (sc-2005). 2-amino-3-methylimidazo [4,5-f] quinoline (IQ) was purchased from Toronto Research Chemicals. Dimethyl sulfoxide (DMSO), methanol, and glacial acetic acid were purchased from Sigma. AFB1 was dissolved in DMSO, while IQ was dissolved in methanol containing 0.1% acetic acid, and is simply referred to as MeOH.
5
Immunohistochemical Analysis of Sciatic Nerve
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Briefly, rats were anesthetized and then perfused with 100 ml of saline solution and 100 ml of 4% paraformaldehyde. Within 15 min after completion of perfusion, the bilateral sciatic nerves were removed and fixed overnight in 4% paraformaldehyde at 4 °C. Subsequently, the bilateral sciatic nerves were transferred into a 30% sucrose solution for at least 1 week. Tissue embedded into the Tissue-Tek OCT compound (Sakura Finetek) was frozen-sectioned. Next, the sciatic nerve sections were stained using anti-CYP1A2 (5 μg/ml, Abcam, ab22717) and anti-S100β(dilution 1:1,000, Abcam, ab52642). Furthermore, ImageJ measured the fluorescence intensity of immunostaining.
6
Regulation of CYP1A1/2 and Lipid Metabolism
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Oligos (60 bp, containing sequences of CYP1A1 and CYP1A2 target sites) and all primer pairs for PCR were synthesized by Biosune Biotechnology Co., Ltd. (Shanghai, China). SYBR Premix Ex Taq and Prime Script RT Reagent Kit were bought from Takara (Dalian, China). Primary antibodies for CYP1A1/2 (ab22717), LXRα (ab176323), CES1 (ab45957) and GAPDH (ab181602) were purchased from Abcam (Cambridge, UK). Primary antibodies for SCD1 (sc-58420) and SREBP1 (sc-365,513) were supplied by Santa Cruz (Heidelberg, Germany). Primary antibody for CES2 (BS5659) was supplied by Bioworld technology, Co., Ltd. (Nanjing, China). The fluorescence-conjugated secondary antibody to rabbit IgG and mouse IgG were purchased from Cell Signaling Technology (Boston, USA). Lansoprazole (Lan) was purchased from Energy Chemical (Shanghai, China). Cholesterol rich diet (D12108C, cholesterol content 1.25%) was bought from Research Diet (New Brunswick, USA). The ELISA kit for pregnenolone detection was purchased from Novus biological (Centennial, USA). Free cholesterol detection kit was bought from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).
7
Quantification of TMAO and Related Metabolites
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Trimethylamine hydrochloride, TMAO, NADPH, magnesium chloride, tris (hydroxymethyl) aminomethane (Trizma® base), Trizma® hydrochloride, n-octylamine, methimazole, L-arginine and formic acid ( ≥ 95%) were purchased from Sigma-Aldrich (St. Louis, MO). Deuterated internal standard (d9-trimethylamine N-oxide) was purchased from Cambridge Isotopes (Cambridge, MA, USA). Optima LC–MS grade water, acetonitrile, and methanol was purchased from Fisher Scientific (Pittsburgh, PA). Taqman® primers used for mRNA quantification were purchased from Applied Biosystems (Foster City, CA). All fluorescent antibodies were purchased from Abcam (Cambridge, MA) (Codes: ab2769, ab8226, ab22717, ab126790, ab195627, ab186693 and ab175774).
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