Human TNBC cell lines MDAMB468, HCC1806, HCC1569, Hs578t, BT549, MDAMB231, and HCC1937 (Wu et al., 2015 (link)) were procured from American Type Culture Collection (ATCC, Manassas, VA), revived from early passage liquid nitrogen vapor stocks as required and maintained at 37°C in 5% CO2 and 95% humidity. All cells were authenticated via short tandem repeat testing. No mycoplasma contamination was noted. DAPT and GANT61 were procured from Sigma-Aldrich, St. Louis, MO, and Selleck Chemicals, Houston, TX. For Western blot, immunoprecipitation, immunofluorescence and immunohistochemistry, anti-ALDH1A, anti-Nanog, anti-Oct4, anti-KLF4, anti-c-MYC, anti-NICD, anti-JAGGED, and anti-Ki67 antibodies were purchased from Cell Signaling Technology, Beverly, MA. Antibodies anti-GLI1, anti-Sox2, anti-SHH, anti-FOXM1, and anti-HES1 were procured from Santa Cruz Biotechnology Inc Anti-HEY1 was purchased from ABclonal Technology, Woburn, MA. Mouse monoclonal β-Actin was procured from Sigma-Aldrich, St. Louis, MO. Horseradish peroxidase conjugated goat anti-rabbit IgG, goat anti-mouse IgG, and donkey anti-goat IgG were purchased from Sigma-Aldrich, St. Louis, MO. 3-(4,5-Dimethylthiazol-2-yl)–2,5-diphenyltetrazolium bromide (MTT) were procured from Sigma-Aldrich, St Louis, MO. Chemiluminescent peroxidase substrate was procured from GE Healthcare, UK.
Anti gli1
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-GLI1 is a primary antibody that recognizes the GLI1 protein, a transcription factor involved in the Hedgehog signaling pathway. This antibody can be used for applications such as Western blotting and immunohistochemistry to detect and study the expression of GLI1 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti shh, by Santa Cruz Biotechnology (9 mentions)
Anti smo, by Santa Cruz Biotechnology (4 mentions)
Anti e cadherin, by Santa Cruz Biotechnology (3 mentions)
Anti ptch1, by Santa Cruz Biotechnology (3 mentions)
Anti sox2, by Santa Cruz Biotechnology (2 mentions)
Anti vegf, by Abcam (2 mentions)
Anti cd34, by Santa Cruz Biotechnology (2 mentions)
Confocal laser scanning microscope, by Olympus (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Merck Group (1 mentions)
Anti c myc, by Cell Signaling Technology (1 mentions)
Gant61, by Merck Group (1 mentions)
Anti oct4, by Cell Signaling Technology (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Anti nanog, by Cell Signaling Technology (1 mentions)
Anti nicd, by Cell Signaling Technology (1 mentions)
Anti klf4, by Cell Signaling Technology (1 mentions)
Donkey anti goat igg, by Merck Group (1 mentions)
21 protocols using anti gli1
1
Characterization of TNBC Cell Lines
2
Protein Expression Analysis in Kidney Tissues
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Cells and kidney tissues were washed with PBS and lysed in the M-PER mammalian protein extraction reagent with protease inhibitor cocktail (Thermo Fisher Scientific Inc., San Jose, CA, USA). Proteins were separated with 8–15% SDS-PAGE and then were transferred onto a nitrocellulose membrane (Millipore, Madrid, Spain) by electroblotting. The membrane was blocked for 1 hour at room temperature and then was incubated overnight at 4 °C with anti-Shh, anti-E-cadherin, anti-Smad2, anti-Smo, anti-Gli-1 (1:1000, Santa Cruz biotechnology, Santa Cruz, CA, USA), anti-fibronectin (R&D system Inc. Minneapolis, MN, USA), anti-Bax, anti-Bcl-2, anti-TGF-β1 (1:1000, Cell Signaling Technology, Beverly, MA, USA), and α-SMA (1:1000 Abcam Inc. Cambridge, MA, USA) primary antibodies. Subsequently, the membranes were stained with horseradish peroxidase-conjugated goat anti-rabbit or mouse immunoglobulin G (1:2,000, Santa Cruz biotechnology, Santa Cruz, CA, USA). The immunoreactive bands were detected by chemiluminescence (enhanced chemiluminescence; BioFX Laboratories Inc., Owings Mills, Maryland, USA). GAPDH (1:2,000, Santa Cruz biotechnology, Santa Cruz, CA, USA) was used as an internal control.
3
Immunofluorescence Staining of Paraffin Sections
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Paraffin sections for IF were stained with anti-Shh (1:100, Santa Cruz Biotechnology, USA), anti-Gli1 (1:100, Santa Cruz Biotechnology, USA), anti-VEGF (1: 100, Abcam, UK) and anti-CD34 (1: 100, Santa Cruz Biotechnology, USA) overnight at 4°C. Then the sections were washed with PBS and then incubated with the secondary antibody FITC-labeled goat anti-mouse antibody (1:50, ZSGB-BIO, China) at 37°C for 1 h. Next, the cellular nuclei were stained with propidium iodide (PI) solution for 5 min in the dark. Finally, the images were captured using a confocal laser scanning microscope (Olympus, Japan) and the mean fluorescence density was analyzed by the Image-Pro Plus 6.0 software (Media Cybernetics, USA).
4
Immunohistochemical analysis of Shh/Ptch1/Gli1 in lung
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Lung sections were incubated for 18 h at 4°C with one of the following primary antibodies: anti-Shh (1:200; Santa Cruz Biotechnology), anti-Ptch1 (1:100; Santa Cruz Biotechnology), and anti-Gli1 (1:200; Santa Cruz Biotechnology). The secondary biotinylated anti-immunoglobulin antibody and horseradish peroxidase-conjugated streptavidin were then sequentially added and detected using the Polink-2 HRP Plus Rabbit Detection System (Beijing Zhongshan Goldebridge Biotechnology Co., Ltd, Beijing, China). Brown 3,3′-diaminobenzidine (DAB) staining (DAB Detection Kit; Beijing Zhongshan Goldebridge Biotechnology Co., Ltd) indicated the presence of Shh/Ptch1/Gli1-positive cells in the membrane/cytoplasm/nuclei. In negative control slides, the primary antibody was replaced with PBS. Three different sections were used from each lung specimen, and five random fields were selected from each section. Optical density (OD) per area was analyzed using Image-Pro Plus 6.0 system (Media Cybernetics, Inc., Rockville, MD, USA). OD calculations were performed blindly by two pathologists from the Second Xiangya Hospital.
5
Tissue Morphogenesis and Signaling Pathway Analysis
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Sections were prepared as described previously [2 (link)]. Sections were stained with haematoxylin and eosin (H&E) for the tissue morphogenetic study. Immunohistochemistry analyses were performed following the manufacturer’s instructions. The primary antibodies used were anti-Wnt10b, anti-BMP4, anti-Gli1, anti-SHH, anti-SOX2, anti-β-catenin and anti-OSR2 (Santa Cruz). Images were taken using a microscope (Olympus BX43F) with an attached Olympus DP72 digital camera system.
6
Western Blot Analysis of Hedgehog Pathway
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Fifty µg of protein (determined by Bio-Rad Protein Assay; Bio-Rad, Hercules, California, USA) was loaded on SDS-polyacrylamide gel, transferred to a nitrocellulose membrane and blocked in 5% milk. Primary antibodies (diluted 1∶250) for Shh and ERα were the same as for the immunofluorescence experiment, additionally goat polyclonal anti-Ptch1 (Santa Cruz Biotechnology, sc-6147) and rabbit polyclonal anti-Gli1 (Santa Cruz Biotechnology, sc-20687) were used. Actin (Santa Cruz Biotechnology, sc-1616, goat polyclonal, diluted 1∶500) was used as loading control. After washing, membranes were incubated with the appropriate secondary HRP-conjugated antibody (Santa Cruz Biotechnology). Proteins were visualized using Super Signal West Pico and Femto reagents (Thermo Fisher Scientific, Waltham, Massachusetts, USA).
7
Shh Pathway Activation in Ischemic Cortex
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The proteins were isolated from the ischemic cortex tissue using lysis buffer (Beyotime, China), and protein concentrations were examined using a BCA protein assay kit (Beyotime, China). The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidenedifluoride (PVDF) membranes. After blocking with 5% skimmed milk, the membranes were incubated with primary antibodies: anti-Shh, anti-Ptch, anti-Smo, anti-Gli1, anti-CD34 (1:1000, Santa Cruz Biotechnology, USA), and anti-VEGF (1:1000, Abcam, UK) overnight at 4 °C. After rinsing four times in TBST buffer for 5 min, the membranes were incubated with secondary antibodies (1:20000, ZSGB-BIO, China) at room temperature for 2 h and then treated with ECL reagent (ThermoFisher, USA) to detect protein expression levels. The protein bands were quantitatively analyzed using the Image J software (Rawak Software Inc., Germany).
8
Immunohistochemical Analysis of Tumor Samples
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After antigen retrieval and blocking (Supplemental Table S1 ), tumor sections were incubated overnight at 4°C with anti-pimonidazole (1/400, Hypoxyprobe, Massachusetts, USA), anti-caspase-3 (ready to use, Biocare Medical, Concord CA, USA) or anti-CD31 (1/25, Dianova, Hamburg, Germany), anti-GLI1 (1/50, Santa-Cruz), anti-GLI2 (1/1000, Rockland), anti-PTCH1 (1/300, Santa Cruz); or for 30 min at room temperature with anti-Ki67 (ready to use, Thermo Scientific). Appropriate secondary antibodies followed by 3.3′-diaminobenzidine (DAB) substrate (DAKO, Glostrup, Denmark) were used to visualize antigen presence.
Quantification of Ki67 was performed by counting the number of Ki67 positive nuclei in the tumor tissue. Mean vessel density (MVD) was assessed as the number of blood vessels (CD31+) per field for 10 high-power fields per tissue specimen. Tumor hypoxic fraction was determined as the percentage of cytoplasmic pimonidazole positive cells. The apoptotic fraction was determined by assessing the number of caspase-3 positive cells per tissue section.
Quantification of Ki67 was performed by counting the number of Ki67 positive nuclei in the tumor tissue. Mean vessel density (MVD) was assessed as the number of blood vessels (CD31+) per field for 10 high-power fields per tissue specimen. Tumor hypoxic fraction was determined as the percentage of cytoplasmic pimonidazole positive cells. The apoptotic fraction was determined by assessing the number of caspase-3 positive cells per tissue section.
9
Western Blot Analysis of Gli1 Protein
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Forty-eight hours after transfection, cells were harvested and lysed in a solution containing Tris-HCl pH 7.6, 50mM deoxycholic acid, sodium salt 1%, NaCl 150mM, NP40 1%, EDTA 5mM, NaF 100mM and protease inhibitors. Lysates were separated on SDS-PAGE and immunoblotted using standard procedures. Anti-Gli1 (Santa Cruz Biotechnology), anti-actin (Santa Cruz Biotechnology) and HRP-conjugated secondary antisera (Santa CruzBiotechnology) were used. Bands were visualized by EZ-ECL detection reagents.
10
Immunofluorescence Analysis of Cilia and GLI1
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Cells were seeded on glass coverslips, allowed to attach for 48 h, and then fixed for 10 min in 4% paraformaldehyde, washed in PBS, incubated for 5 min in methanol, and then incubated over-night at 4 °C with rabbit polyclonal anti-Arl13b (Proteintech, Rosemont, IL, USA; Cat #17711-1-AP, RRID: AB_2060867) and anti-GLI1 (Santa Cruz Biotechnology, CA, USA; Cat #sc-515781). The appropriate secondary antibodies were incubated for 45 min at room temperature (anti-mouse Alexa Fluor 555 (Invitrogen, Waltham, MA, USA; Cat #A21422) or anti-Rabbit Fitc (Sigma-Aldrich, St. Louis, MO, USA, Cat #F9887). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI).
Unspecific signal was evaluated for each antibody using a control condition without primary antibody and with a non-specific antibody. Images were acquired using the Zeiss Axio microscope (Zeiss Laboratories, White Plains, NY, USA). The proportion of ciliated cells was determined across multiple fields of view for each condition.
Unspecific signal was evaluated for each antibody using a control condition without primary antibody and with a non-specific antibody. Images were acquired using the Zeiss Axio microscope (Zeiss Laboratories, White Plains, NY, USA). The proportion of ciliated cells was determined across multiple fields of view for each condition.
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