To verify the expression of HIF-1α and VEGF proteins, the total protein in the tissue samples was homogenized and lysed with ice-cold RIPA buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1% NP40, protease, and phosphatase inhibitor). Protein extracts were separated using 10% polyacrylamide gel and were electro-transferred to nitrocellulose membranes. After blocking with 5% milk in TBST buffer (10 mmol/L Tris-HCl, 150 mmol/L NaCl, and 0.05% Tween-20), the membranes were probed with primary antibodies: HIF-1α (NB100-449, Novus Biologicals, Littleton, CO, USA); VEGF (ab46154, Abcam, CA, MA, USA); and actin (A2066, Sigma-Aldrich, St. Louis, MO, USA), followed by incubation with HRP-conjugated secondary antibody (T6778, Sigma-Aldrich, St. Louis, MO, USA). The target bands were visualized using enhanced chemiluminescence reagent (Amersham Biosciences), and the band images were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
T6778
Manufactured by Merck Group
Sourced in United States, Denmark
T6778 is a laboratory equipment product. It is a multi-functional device designed for various scientific applications. The core function of T6778 is to facilitate precise measurements and analysis within a controlled laboratory environment. Detailed specifications and intended use are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab53707, by Abcam (2 mentions)
Ab92547, by Abcam (2 mentions)
Hrp conjugated secondary antibody, by Merck Group (1 mentions)
Enhanced chemiluminescence reagent, by Cytiva (1 mentions)
Nb100 449, by Novus Biologicals (1 mentions)
Ab46154, by Abcam (1 mentions)
Hif 1α, by Novus Biologicals (1 mentions)
Ix51 fluorescence microscope, by Olympus (1 mentions)
Xm10 digital camera, by Olympus (1 mentions)
Hpa053669, by Merck Group (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Wheat germ agglutinin (wga), by Thermo Fisher Scientific (1 mentions)
Chamber slide, by Thermo Fisher Scientific (1 mentions)
Tcs5 laser scanning confocal microscope, by Leica (1 mentions)
Vectashield dapi, by Vector Laboratories (1 mentions)
Lsm 510 confocal microscope, by Leica (1 mentions)
T8787, by Merck Group (1 mentions)
P1379, by Merck Group (1 mentions)
A11001, by Thermo Fisher Scientific (1 mentions)
F3648, by Merck Group (1 mentions)
5 protocols using t6778
1
Western Blot Analysis of HIF-1α and VEGF
2
Immunofluorescence Analysis of SIRT7 and Cell-Cell Adhesion Proteins
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Wild-type MGHU3, 5637 and J82, sh-scramble/CTRL and/or sh-SIRT7 cells were seeded on cover slips at 20,000 cells/well, overnight. Briefly, cells were fixed in methanol during 10 min and then blocked with 5% bovine serum albumin (BSA) during 30 min. After overnight SIRT7 (1:500, HPA053669, Sigma-Aldrich), ECAD (1:150, #3195, Cell Signaling Technology) and/or NCAD (1:50, #13116, Cell Signaling Technology) incubation at RT, cells were incubated with s ondary antibody anti-rabbit IgG-TRITC (1:500, T6778, Sigma-Aldrich) during 1 h at RT. Finally, after 1× PBS wash, cells were stained with 4’,6-diamidino-2-phenylindole (DAPI) (AR1176, BOSTER Biological Technologies (Pleasanton, CA, USA) r in mounting medium. Pictures were taken on a IX51 fluorescence microscope (Olympus, Tokyo, Japan) equipped with an Olympus XM10digital camera using CellSens software.
3
Immunofluorescence Staining of Transfected Cells
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Transfected cells were grown on the glass coverslips or chamber slides (Nunc, Roskilde, Denmark) for 24 to 48 h. Cell membranes were visualized by staining cells with 7 µg/mL of fluorescein isothiocyanate (FITC) conjugated Wheat germ agglutinin (WGA; Molecular Probes, Eugene, OR, USA) in PBS. After that, cells were fixed with 3,7–4% paraformaldehyde in PBS for 10 to 20 min. Fixed cells were permeabilized with 0.2% Triton X-100 in PBS for 3 min, blocked with PBS containing 2% of BSA and 0.2% Tween-20, and incubated first with anti-core rabbit serum diluted 25 to 250 in the Dako Cytomation Antibody Diluent (Dako Cytomation, Glostrup, Denmark) and then up to 1 h with FITC- or TRITC-conjugated goat anti-rabbit IgG (F9887 or T6778, respectively, Sigma). Finally, DAPI was added at 1 µg/mL for 5 min. All incubations were done at 20 °C. Slides were then mounted with Prolong Gold Antifade Reagent (Molecular Probes), covered with cover slips and read on a Leica TCS5 laser scanning confocal microscope (Leica, Wetzlar, Germany). Images were scanned and quantified using ImageJ software.
4
Immunocytochemical Analysis of Cellular Phenotypes
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Cells grown on 13 mm coverslips were fixed in 4% paraformaldehyde for 10 minutes and then washed with 1× phosphate buffered saline (PBS). Glycine (100 mM) was added to quench any remaining fixative. Cells were then permeabilized in 0.1% Triton-X100 (T8787, Sigma UK) in 1× PBS for 30 minutes and washed twice in PBS-0.2% Tween 20 (P1379, Sigma UK) and once further in 1× PBS. Cells were then either stained with H&E to assess cellular morphology or using immunocytochemical techniques for epithelial and mesenchymal markers. Primary antibodies were incubated at 4oC overnight (rabbit anti-human cytokeratin 17 Abcam Ab53707, Rabbit anti-human vimentin Abcam Ab92547), followed by fluorophore-conjugated secondary antibody (goat anti-rabbit TRITC conjugated Sigma T6778) at 1:100 dilution in 5% bovine serum albumin PBS-0.2% Tween 20 incubated for 90 minutes in the dark. Negative controls were performed with secondary only antibodies and matched IgG isotype negative controls to identify if there were nonspecific binding or background autofluorescence. Coverslips were mounted on slides with DAPI Vectashield (H1200, Vector Labs, Burlingame, California, USA), and images were captured with a Leica LSM 510 confocal microscope.
5
Immunofluorescence Analysis of Cell Markers
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Cells (1 × 104 cells/cm2) were seeded onto coverslips for 24 hours and fixed in 4% paraformaldehyde. Subsequently cells were washed and quenched with 100 mM glycine before permeabilisation using 0.1% Triton X-100 and blocking with 5% bovine serum albumin (BSA). Primary antibodies (α-SMA (Ab5694, Abcam), β-tubulin (T4026, Sigma), collagen I (C2456, Sigma), cytokine 17 (Ab53707, Abcam), E-cadherin (610181, BD Bioscience), fibronectin (F3648, Sigma), vimentin (Ab92547, Abcam & M7020, Dako) and ZO-1 (33–9100, Zymed)) were added and incubated overnight at 4° C in 5% BSA. Cells were washed with 0.2% tween-20 in PBS and then incubated with secondary antibodies (anti-rabbit TRITC (T6778, Sigma) and anti-mouse Alexafluor 488 (A11001, Molecular Probes)) for 60 mins in 5% BSA. Cover slips were washed and mounted in mounting media containing DAPI (Vectra Shield). Images acquired using a Leica TCS SP2 UV confocal microscope.
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