Investigations on human patient samples have been conducted in accordance with the local ethics committees (ethics committees of the Canton Thurgau and Eastern Switzerland) and the Declaration of Helsinki and informed consent was obtained from all subjects. Formalin-fixed and paraffin-embedded lung cancer tissue sections were provided by Achim Fleischmann (Kantonspital Münsterlingen, Switzerland). Heat-induced antigen retrieval was performed in sodium citrate pH 6,0 (anti-CYP11B1 antibody) or Tris-EDTA pH 9,0 (anti-11β-HSD1 antibody) buffer. Endogenous peroxidase was blocked with 1% hydrogen peroxide (Sigma-Aldrich). The sections were stained using a rabbit anti-human 11β-HSD1 antibody (ab39364, Abcam), rabbit anti-human CYP11B1 antibody (HPA056348, Sigma) or rabbit isotype control, and a biotin-labelled secondary antibody. Vectastain ABC-kit (Vector Laboratories, Burlingame, CA, US) was used to convert the substrate DAB (Roche) into a brown colored product. Nuclei were visualized with hematoxylin (Roth).
Ab39364
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab39364 is a lab equipment product offered by Abcam. It is a device used for scientific research and analysis purposes. The core function of this product is to facilitate specific laboratory procedures or experiments. Further details about its intended use or performance characteristics are not available.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine (dab), by Roche (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Ab17721, by Abcam (1 mentions)
H3k4me2 antibody, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Imagequant las 3000, by GE Healthcare (1 mentions)
Ripa buffer, by Fujifilm (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Ab8227, by Abcam (1 mentions)
4 protocols using ab39364
1
Immunohistochemical Analysis of 11β-HSD1 and CYP11B1 in Lung Cancer
2
Antibody Protocol for PRDM16, LSD1, CTBP1 ChIP and WB
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PRDM16 antibody for immunoprecipitation, ChIP, and Western blot was from R&D Systems (AF6295). LSD1 antibody for Western blot was from Abcam (ab17721), and LSD1 antibody for ChIP was from EMD Millipore (05-939). CTBP1 antibody was from Abcam (ab129181). H3K4me1 antibody was from Diagenode (C1540194). H3K4me2 antibody was from EMD Millipore (07-030). HSD11B1 antibody was from Abcam (ab39364).
3
Quantitative Western Blot Analysis
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Total hepatic protein extract was obtained as previously described (Lazaro et al., 2013 (link)). The protein was separated by SDS–PAGE and transferred to a 0.2 μm PVDF membrane. The primary antibodies used were: 11β-HSD1 (rabbit polyclonal antibody, Abcam, catalog # ab39364; being blocked with 11β-HSD1 peptide, #ab101097, San Francisco, CA), ACS2 (Abcam, catalog # ab229958, San Francisco, CA, United States), and β-actin (ACTB, Cell signaling, Danvers, MA, United States). Goat anti-rabbit HRP was used as a secondary antibody. Finally, chemiluminescence emitted by the immunoreactive protein was detected by an ECL advance agent, and visualized with a MicroChemi Imaging system and qualified by a densitometry using AlphaEaseFC (ProteinSample, CA, United States). Relative protein levels were normalized by ACTB.
4
Western Blot Analysis of Steroid Enzymes
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The kidney, adrenal gland, gastrocnemius muscle, and liver were homogenized in RIPA buffer (Wako Pure Chemicals, Osaka, Japan). In brief, the extracted protein were boiled in EzApply Buffer (Atto, Tokyo, Japan) for 3 min. Sodium dodecyl sulfate-treated proteins (20 μg) were separated by electrophoresis on a 12.5 % polyacrylamide gel (Atto, Tokyo, Japan) and transferred onto membranes. (Atto, Tokyo, Japan) The membranes were incubated overnight at 4°C with primary antibodies against Hsd11b1 (ab39364; Abcam, Cambridge, UK), Hsd11b2 (14192-1-AP; Proteintech, Rosemont, USA), Srd5a1 (ab110123; Abcam, Cambridge, UK), and beta-actin (ab8227; Abcam, Cambridge, UK) [26] (link). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Immunoreactive bands were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA). The intensities of specific chemiluminescence bands were analyzed using a Lumino image analyzer (ImageQuant LAS 3000; GE Healthcare, Amersham, UK).
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