Ncl l pgr 312

Manufactured by Leica

Sourced in United Kingdom, United States

The NCL-L-PGR-312 is a lab equipment product developed by Leica. It is a specialized device designed for scientific and research applications. The core function of this product is to perform specific tasks within a laboratory environment. However, due to the need to maintain an unbiased and factual approach, a detailed description of the intended use or application of this product cannot be provided.

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Lab products found in correlation

Sc 2313, by Santa Cruz Biotechnology (2 mentions) H 300, by Santa Cruz Biotechnology (2 mentions) Protease inhibitor, by Roche (2 mentions) Ncl l er 6f11, by Leica (2 mentions) Pierce ecl chemiluminescent western blotting substrate, by Thermo Fisher Scientific (1 mentions) Gapdh 0411, by Santa Cruz Biotechnology (1 mentions) Histomount, by Thermo Fisher Scientific (1 mentions) K5007, by Agilent Technologies (1 mentions) Phosphatase inhibitor, by Merck Group (1 mentions) Clarity western enhanced chemiluminescence substrate, by Bio-Rad (1 mentions) β actin clone ac 15, by Merck Group (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Ripa buffer, by Merck Group (1 mentions) Protease inhibitor, by Merck Group (1 mentions) Bradford reagent, by Bio-Rad (1 mentions) Prolong gold antifade with dapi, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 anti mouse antibody, by Thermo Fisher Scientific (1 mentions) Triton x 100, by Merck Group (1 mentions) Sc 2005, by Santa Cruz Biotechnology (1 mentions) Herceptest, by Agilent Technologies (1 mentions)

12 protocols using ncl l pgr 312

Quantification of Phosphorylated Human Progesterone Receptor

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Western blotting was carried out as previously described [30 (link)]. The primary antibodies for hPR (NCL-L-PGR-312, Leica Biosystems, USA), phospho-hPR (Ser294) (MA1414, ThermoFisher Scientific, RSA) and GAPDH (0411 sc-47724 Santa Cruz Biotechnology, Inc, USA) were diluted 1:1000, 1:100 and 1:4000, respectively. Membranes were washed and incubated for 1 h with the secondary goat-anti-mouse (sc-2313, Santa Cruz Biotechnology, Inc, USA) antibody (1:5000). Thereafter, the membranes were washed again and incubated with Pierce ECL-chemiluminescent western blotting substrate (Thermo Scientific, USA). Proteins were visualized by autoradiography using Amersham Hyperfilm™ MP high performance autoradiography film (AEC-Amersham, RSA). For consecutive detection steps, membranes were stripped as previously described [30 (link)]. Densitometric quantification of film was carried out using ImageJ™ Software (NIH, USA).

Enfield K., Dlamini S., Avenant C., Kuipa M, & Hapgood J.P. (2020). Maraviroc, tenofovir disoproxil fumarate and dapivirine, activate progesterone receptor B in the absence of progestogens. Biochemical and biophysical research communications, 533(4), 1027-1033.

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Immunohistochemical Staining of TFF, ER, and PR

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Whole sections of formalin-fixed, paraffin-embedded tissue were deparaffinised, rehydrated and incubated in 0.5% hydrogen peroxide for 10 min and trypsin for 10 min or 10 mM citrate buffer (pH 6.0) in a pressure cooker for 1 min. Endogenous biotin was blocked with the Dako Cytomation. Sections were incubated with monoclonal antibodies against TFF3 protein, TFF1 protein (Westley et al. 2005 (link)), oestrogen receptor (NCL-L-ER-6F11; Leica Biosystems, Newcastle Upon Tyne, UK) or progesterone receptor (NCL-L-PGR-312; Leica Biosystems), secondary antibody and avidin-biotin immunoperoxidase complex (Vector Laboratories, Peterborough, Cambridgeshire, UK), developed with diaminobenzidine and counterstained with haematoxylin.

May F.E, & Westley B.R. (2015). TFF3 is a valuable predictive biomarker of endocrine response in metastatic breast cancer. Endocrine-Related Cancer, 22(3), 465-479.

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Western Blot Analysis of ER and PR

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MCF-7 BUS cell lysates from siRNA transfections were subjected to electrophoresis on a 10% SDS-polyacrylamide gel, before transfer of proteins to nitrocellulose membranes (AEC Amersham, RSA). The membranes were then probed with primary antibodies specific to ERα (F-10, Santa Cruz Biotechnology, USA), the PR isoforms (NCL-L-PGR-312, Leica Biosystems, Germany) or the loading control, GAPDH (0411, Santa Cruz Biotechnology, USA), followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody (anti-mouse, sc-2005, Santa Cruz Biotechnology, USA). Proteins were visualized using enhanced chemiluminescence (Biorad, RSA) and a MyECL imager and quantified using ImageJ software (version 1.49).

Perkins M.S., Louw-du Toit R., Jackson H., Simons M, & Africander D. (2022). Upregulation of an estrogen receptor-regulated gene by first generation progestins requires both the progesterone receptor and estrogen receptor alpha. Frontiers in Endocrinology, 13, 959396.

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Immunohistochemical Analysis of Hormone Receptors

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Tissues were paraffin-embedded and subjected to immunohistochemical staining. For this procedure, 4-μm sections were deparaffinized and rehydrated, rinsed in purified water, and treated with 3% H₂O₂ for 15 min at room temperature. After rinsing three times with purified water, they were heated in an autoclave for 1 h with 10 mM citrate buffer and then incubated with primary antibodies against estrogen receptor-α (ERα; EP1 diluted 1:50, Bio SB, CA, USA), ERβ (14C8 diluted 1:100, Abcam, Cambridge, UK), progesterone receptor (PR; NCL-L-PGR-312 diluted 1:50, Leica Biosystems, Benton Lane, UK), and androgen receptor (AR; Clone SP107 diluted 1:50, ZECA, CA, USA). After the addition of the appropriate secondary IgG antibody, sections were incubated with DAB (K5007, Dako, Denmark) and counterstained with hematoxylin and eosin (H&E; 1.05174, Merck, MA, USA). Finally, sections were dehydrated in a graded series of ethanol, cleared with xylene, mounted using Histomount (008030, Life Technologies, MD, USA), and coverslips were applied for evaluation by light microscopy.

Lan K.C., Lai Y.J., Cheng H.H., Tsai N.C., Su Y.T., Tsai C.C, & Hsu T.Y. (2020). Levels of sex steroid hormones and their receptors in women with preeclampsia. Reproductive Biology and Endocrinology : RB&E, 18, 12.

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Western Blot Analysis of PR Protein

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Whole cell lysates were prepared in RIPA buffer (Sigma) containing 2 mM sodium orthovanadate, protease inhibitor (1:500, Sigma), and phosphatase inhibitor (1:100, Sigma). Protein was quantified using Bradford reagent (Bio-Rad). Equal amounts of protein were run on 10% SDS-PAGE, transferred to PVDF membrane (Millipore), and probed with antibodies against PR (NCL-L-PGR-312, Leica Biosystems, 1:1000, 2 h at room temperature) and β-actin (clone AC-15, Sigma, 1:10,000, overnight at 4°C). Blots were imaged using Clarity Western enhanced chemiluminescence substrate (Bio-Rad) and GeneSys GBox:XX6 (Syngene).

Fowler A.M., Salem K., DeGrave M., Ong I.M., Rassman S., Powers G.L., Kumar M., Michel C.J, & Mahajan A.M. (2020). Progesterone Receptor Gene Variants in Metastatic Estrogen Receptor Positive Breast Cancer. Hormones & Cancer, 11(2), 63-75.

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Immunofluorescent Localization of PR

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Cells were grown on chamber slides (Lab-Tek II) and then fixed with 3.7% formaldehyde, permeabilized with 0.5% Triton X-100 (Sigma), blocked with 10% goat serum for 1 h at 37°C, and incubated with anti-PR antibody (1:100, NCL-L-PGR-312; Leica Biosystems) overnight at 4°C. Next, the slides were incubated with Alexa Fluor 488 anti-mouse antibody (1:100, Life Technologies) for 1 h at room temperature, mounted with ProLong Gold Antifade with DAPI (Life Technologies), and imaged using confocal microscopy (Leica SP8 STED).

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Protein Extraction and Western Blotting of GR and PR

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Total protein lysates from PBMCs were harvested using .1 M N‐[Tris(hydroxymethyl)‐methyl]‐3‐aminopropanesulfonic acid (TAPS) buffer, pH 9.5, supplemented with protease inhibitors (Roche Applied Science, South Africa) as previously described.⁶⁹Western blotting was carried out as previously described.⁷⁰ Blots were probed with primary antibodies against GR (1:2000; H‐300, Santa Cruz Biotechnology), PR (1:500; NCL‐LPGR‐312, Leica Biosystems) and GAPDH (1:15000; 0411, Santa Cruz Biotechnology). Horseradish peroxidise‐conjugated secondary antibodies, anti‐mouse (1:4000; sc‐2005) and anti‐rabbit (1:10000; sc‐2313), were purchased from Santa Cruz Biotechnology, USA.

Bick A.J., Avenant C., Tomasicchio M., van der Spuy Z, & Hapgood J.P. (2022). Increased HIV‐1 infection in PBMCs treated in vitro with menstrual cycle phase hormones or medroxyprogesterone acetate likely occurs via different mechanisms. American Journal of Reproductive Immunology, 88(6), e13643.

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Protein lysate preparation and western blotting

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Total protein lysates from PBMCs were harvested using 0.1 M N-[Tris(hydroxymethyl)-methyl]-3-aminopropanesulfonic acid (TAPS) buffer, pH 9.5, supplemented with protease inhibitors (Roche Applied Science, South Africa) as previously described^{69 (link)}.
Western blotting was carried out as previously described^{70 (link)}. Blots were probed with primary antibodies against GR (1:2000; H-300, Santa Cruz Biotechnology), PR (1:500; NCL-LPGR-312, Leica Biosystems) and GAPDH (1:15000; 0411, Santa Cruz Biotechnology). Horseradish peroxidise-conjugated secondary antibodies, anti-mouse (1:4000; sc-2005) and anti-rabbit (1:10000; sc-2313), were purchased from Santa Cruz Biotechnology, USA.

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Comprehensive IHC and FISH Assessment of ER, PR, and HER2

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The ER and PR status were evaluated histologically on immunohistochemical (IHC) slides [ER: SP1 (NeoMarkers), PR: NCL-L PGR-312 (Novocastra)]. ER and PR positivity were defined when at least 10% prevalence of malignant cells exhibiting staining characteristics.
HER2 protein overexpression was assessed by IHC method using three different antibodies: RTV-CB11 (Novocastra), C-erbB-2/Her-2/neu SP3 clone (NeoMarkers) and HercepTest (DAKO). Samples were scored using the recommended scoring system for the HercepTest.
HER2 gene amplification was tested with the Inform-HER2 test by Ventana. The updated cut-off value for positive cases is more than six copies of the gene. HER2 was scored positive if the result was 3+; 2+ was considered to be positive only if it was confirmed by fluorescence in situ hybridization (FISH).

Vincze B., Kapuvári B., Udvarhelyi N., Horváth Z., Mátrai Z., Czeyda-Pommersheim F., Kőhalmy K., Kovács J., Boldizsár M., Láng I, & Kásler M. (2015). Serum estrone concentration, estrone sulfate/estrone ratio and BMI are associated with human epidermal growth factor receptor 2 and progesterone receptor status in postmenopausal primary breast cancer patients suffering invasive ductal carcinoma. SpringerPlus, 4, 387.

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Immunohistochemical Labeling Protocol

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Immunohistochemical labeling was performed at the immunohistochemical laboratory of the Department of Pathology, Asan Medical Center. In brief, 4-mm-thick tissue sections were deparaffinized and hydrated in xylene and serially diluted in ethanol. Endogenous peroxidase was blocked by incubation in 3% H₂O₂ for 10 minutes, and then heat-induced antigen retrieval was performed. Primary antibodies were used with a Benchmark autostainer (Ventana Medical Systems, Tucson, AZ, USA) in accordance with the manufacturer’s protocol. Sections were incubated at room temperature for 32 minutes in primary antibodies for PR (1:200, NCL-L-PGR-312, Novocastra, Newcastle upon Tyne, UK), synaptophysin (1:200, DiNona, Seoul, Korea), chromogranin (1:200, DAK-A3, DakoCytomation, Glostrup, Denmark), and Ki-67 (1:100, 7B11, Zymed, San Francisco, CA, USA). The sections were then labeled with an automated immunostaining system and processed with an iView DAB detection kit (Benchmark XT, Ventana Medical Systems). Immunostained sections were lightly counterstained with hematoxylin, dehydrated in ethanol, and cleared in xylene. Immunoreactivity was interpreted by light microscopic examination and independently evaluated by two pathologists, coauthors of this study (S.J.K. and S.M.H.), who were blind to the clinicopathologic information.

Kim S.J., An S., Lee J.H., Kim J.Y., Song K.B., Hwang D.W., Kim S.C., Yu E, & Hong S.M. (2017). Loss of Progesterone Receptor Expression Is an Early Tumorigenesis Event Associated with Tumor Progression and Shorter Survival in Pancreatic Neuroendocrine Tumor Patients. Journal of Pathology and Translational Medicine, 51(4), 388-395.

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