Cytoblocks were prepared for ICC and DakoEnVision™ Flex+ (K8012; Dako, Glostrup, Denmark) was applied for ICC and IHC staining as previously described [43 (link)]. After deparaffinization, unmasking epitopes and blocking of peroxidase, the slides were then incubated with the following reagents: rabbit monoclonal antibody against human MPC1(HPA045119, Sigma-Aldrich, St. Louis, MO, USA, diluted 1:500) and MPC2(ab111380, abcam, Cambridge, UK, diluted 1:500) at 4°C overnight. Negative controls were produced using the same concentration of non-immune rabbit IgG instead of the rabbit antibody to human MPC1 and MPC2.
Ab111380
Manufactured by Abcam
Sourced in United Kingdom
Ab111380 is a laboratory equipment product. It is a component used in scientific research and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Ab47010, by Abcam (2 mentions)
Hpa045119, by Merck Group (1 mentions)
Dako envision flex, by Agilent Technologies (1 mentions)
Ab104836, by Abcam (1 mentions)
α tubulin antibody t9026, by Merck Group (1 mentions)
Ab2302, by Cell Signaling Technology (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Annexin 5, by Thermo Fisher Scientific (1 mentions)
Lncap, by American Type Culture Collection (1 mentions)
Image pro plus version 5, by Media Cybernetics (1 mentions)
Brp44 antibody, by Abcam (1 mentions)
Anti brp44 l antibody, by Abcam (1 mentions)
4 protocols using ab111380
1
Immunocytochemistry and Immunohistochemistry of MPC1/MPC2
2
Metabolic Enzyme Expression Analysis
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Cells were firstly lysed in RIPA buffer supplemented with a cocktail of protease inhibitors. Cell lysates were centrifuged at 13000 g for 10 minutes at 4°C before aliquots of 20 μg proteins were resolved on SDS-PAGE, transferred onto polyvinylidenedifluoride transfer membrane(PVDF), and blotted for targeting antibodies:MPC1(HPA045119, Sigma-Aldrich, St. Louis, MO,USA, dilution 1:1000), MPC2(ab111380, abcam, Cambridge, UK, dilution 1:1000), GLUT1(Rabbit mAb#12939, cell signaling technology, Leiden, Netherlands, dilution 1:1000), HK2 (ab104836, abcam, Cambridge, UK, dilution 1:1000), LDHA (ab47010, abcam, Cambridge, UK, dilution 1:1000) and α-Tubulin antibody(T9026, Sigma-Aldrich, St. Louis, MO, USA dilution 1:3000).
3
Prostate Cancer Cell Line Cultivation and Metabolic Marker Analysis
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The prostatic cancer cell line LNCaP was obtained from ATCC (American Type culture collection, USA) and maintained in our laboratory for the study. Cells were cultured in RPMI 1640 (Gibco™, 32404-014), supplemented with 10% fetal bovine serum (Gibco™, 10500-064) with or without 4mM L-glutamine (Gibco™, 25030-081), 100 U/ml of penicillin and 100ug/ml streptomycin (15140122, Life Technologies) at 37°C, and 5% CO2. Anti-PDH antibody (cell signaling, C54G1), anti-Pyruvate Dehydrogenase E1-alpha subunit antibody (ab110334), monoclonal anti-GAPDH antibody (sigma, AB2302), anti-GLUT1 antibody (cell signaling, D3J3A), Anti-LDHA antibody (abcam, ab47010), anti-HK2 (abcam, ab104836), anti-GLS1 (sigma, WH0002744M1-100UG), anti-GLUD1 (Novus Biologicals, NBP1-54961), anti-MPC1 (Novus Biologicals, NBP1-91706) and anti-MPC2(abcam, ab111380) were applied in the current study. BPTES (SML0601), EGCG (03970590-10MG) and dimethyl-αKG (34963-1) were obtained from Sigma-Aldrich (Sigma-Aldrich Norway AS Oslo, Norway). Annexin-V was purchased from Life technologies (Life technologies, A13199).
4
Immunohistochemical Analysis of BRP44L and BRP44 Proteins
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All tissue samples were fixed with 10% neutral formalin, embedding into paraffin sections (4 µm) after 24 h and were subsequently deparaffinized with dimethylbenzene and rehydrated through a concentration gradient of ethanol, prior to antigen retrieval in a pressure cooker (pH=6.0 sodium citrate acid repair solution, 121°C for 5 min. Samples were then washed in PBS 3 times, and endogenous peroxidase activity was inactivated in 3% hydrogen peroxide solution for 10 min at room temperature. Sections were incubated with an anti-BRP44L antibody (cat no., ab74871; dilution, 1:50; Abcam, Cambridge, UK) and an anti-BRP44 antibody (cat no., ab111380; dilution, 1:50; Abcam) overnight at 4°C. Horseradish peroxidase-tagged antibody (cat no., PV-9000; ready-to-use; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) was added and incubated for 30 min at room temperature, followed by positive staining with diaminobenzidine (0.05%) for 1 min at room temperature, and counterstaining with hematoxylin (0.1%) for 1 min at room temperature. The slides were observed and imaged under a positive optical microscope. The relative protein expression was quantified by Image-Pro Plus version 5.0 software (Media Cybernetics Inc., Rockville, MD, USA) and defined as follows: Density mean=density sum/area sum (15 (link)).
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