Cells were grown on glass coverslips, washed with phosphate-buffered saline (PBS), fixed with 1 or 4% paraformaldehyde (in PBS) for 10 min, and permeabilized with 0.2% Triton X-100 in PBS for 5 min. After blocking with 2% bovine serum albumin (BSA) in PBS for at least 1 h, cells were incubated with primary antibodies, diluted in 2% BSA in PBS for 1 h at room temperature (RT) or overnight at 4°C, washed three times with PBS, and incubated with secondary antibodies in 2% BSA in PBS for 1 h at RT. After immunostaining, cells were washed with PBS and mounted on microscopic slides with VectaShield (VectorLabs) for microscopic analysis. The following primary antibodies were used in this study: anti-calnexin (Abcam, ab22595), anti-calreticulin (ThermoScientific, PA3-900), CREST (ImmunoVision, HCT0100), anti–Ki-67 (Abcam, ab16667), anti-KIF4 (Abcam, ab3815), anti-KIF22 (Abcam, ab222187), anti-LBR (abcam, ab32535), mAb414 (Abcam, ab24609), anti-MAD2 (Bethyl Laboratories, A300-301A), anti-topoisomeraseII (Abcam, ab109524), anti–α-tubulin (Sigma, T5168), and anti–γ-H2AX (Millipore, 05-636-l). The antibody against human SUN1 has been previously described (Sosa et al., 2013 (link)). For secondary antibodies, Alexa Fluor–conjugated antibodies (goat, 1:300; Life Technologies) were used.
Ab32535
Manufactured by Abcam
Sourced in United Kingdom
Ab32535 is a lab equipment product. It is a tool designed for use in scientific research and laboratory settings. The core function of this product is to perform a specific task or measurement, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab32536, by Abcam (2 mentions)
Ab3815, by Abcam (1 mentions)
Ab109524, by Merck Group (1 mentions)
Ab16667, by Abcam (1 mentions)
Anti calreticulin, by Thermo Fisher Scientific (1 mentions)
Hct0100, by Abcam (1 mentions)
T5168, by Merck Group (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Anti γh2ax, by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Ab112543, by Abcam (1 mentions)
Ab51608, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
Ab8933, by Abcam (1 mentions)
Ab32566, by Abcam (1 mentions)
Ab8926, by Abcam (1 mentions)
Ab108937, by Abcam (1 mentions)
Ab8805, by Abcam (1 mentions)
Ab182858, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
6 protocols using ab32535
1
Immunostaining of Cellular Components
2
Quantitative Western Blot Analysis of THBS2, HIF1, and Lamin B
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Total proteins were extracted from CT26 cells using RIPA buffer. The extracted proteins were separated on a 10% sodium dodecyl sulphate–polyacrylamide gel and transferred onto a PVDF membrane. To prevent non-specific binding, the membrane was blocked with 5% milk at room temperature for 1 h. Subsequently, it was incubated with primary antibodies against THBS2 (1:1,000; ab112543; Abcam, Cambridge, UK), HIF1 (1:500; ab51608; Abcam, Cambridge, UK), lamin B (1:500; ab32535; Abcam, Cambridge, UK) and GAPDH (1:1000; ab8245; Abcam, Cambridge, UK) overnight at 4 °C. The following day, the membrane was washed thrice with TBST and incubated with secondary antibodies (1:2000) for 1 h at room temperature. After the membrane was washed thrice with TBST, protein bands were visualised using an enhanced chemiluminescence reagent. The band intensities were measured using the UN-SCAN-IT gel analysis software, and the expression of target proteins was normalised to that of GAPDH.
3
Molecular Profiling of Ovarian Cancer Cells
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A2780 and SKOV3 cells transfected 48 h later were collected and lysed by RIPA (Beyotime Biotech, China). The lysates were then subjected to fractionation for extracting total protein of the whole cells and nuclear proteins. The proteins were then transferred to PVDF membranes. After being blocked in 5% non-fat milk, the membranes received cultivation (4° C) with primary antibodies targeting Notch2 (1:200; rabbit polyclonal to Notch2: ab8926, Abcam, Cambridge), DNA-PK (1:500; ab32566, Abcam, Cambridge), Hes1 (1:500; ab108937, Abcam, Cambridge), p-Akt (1:500; ab8933, Abcam, Cambridge), Akt (1:1000; ab8805, Abcam, Cambridge), Bcl-2 (1:500; ab182858, Abcam, Cambridge), Bax (1:500; ab32503, Abcam, Cambridge), NF-κB p65 (1:500; ab32536, Abcam, Cambridge), Lamin B (1:1000; ab32535, Abcam, Cambridge) and β-actin (rabbit polyclonal to β-actin: AC026, ABclonal, USA). After washing by Tris-buffered saline containing 0.1% Tween-20 (TBS-T), the membranes got 1 h of probing with the second antibody goat anti-rabbit IgG H&L (HRP; ab6721, Abcam, Cambridge) at 25° C. Subsequent membrane development were performed using the ECL kit (Cat.No.P0018S, Beyotime Biotech, China). Fusion FX7 Spectra (Vilber, France) were used to determine immunoreactivity.
4
Immunofluorescence Characterization of hSGCs
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Isolated hSGCs were identified using cytokeratin 8 (CK8) and α-smooth muscle actin (SMA) immunofluorescence assays. Primary cells were harvested, resuspended in DMEM-F12 and then placed 2x105 cells into 24-well plates with slides (14 mm in diameter; Costar; Corning, Inc.) at 37˚C for 24 h. Next, cells attached to slides were fixed with 4% Paraformaldehyde at room temperature for 30 min, treated with 0.5% Triton X-100 (Beyotime Institute of Biotechnology) at room temperature for 5 min, then incubated with antibodies against CK8 (1:100; cat. no. ab53280; Abcam) and α-SMA (1:200; cat. no. ab32535; Abcam) at 4˚C overnight. This was followed by incubation with secondary goat anti-mouse/rabbit IgG antibody labeled with Alexa Fluor 594/488 (both 1:200; cat. no. ab150113; Abcam) at 37˚C for 1 h. Cell nuclei were stained with DAPI (10 µM) at 37˚C for 10 min. Images of the stained cells were captured using a fluorescence microscope (magnification, x20 and x400; Lionheart LX; BioTek Instruments Inc.).
5
Immunofluorescence Staining Protocol
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Antibodies and dilutions for immunofluorescence were, as primary antibodies, mouse αCLIMP63 (dilution 1:1000G1/296, ABS669-0100 Enzo Life Sciences, Farmingdale, NY, USA), rabbit αRTN4 (dilution 1:300, ab47085, Abcam, Cambridge, UK), rabbit αLBR (dilution 1:300, ab32535, Abcam, Cambridge, UK), and as secondary antibodies goat αRabbit AlexaFluor488 (1:1000, IgG/A-11034, Molecular Probes/Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) and donkey αMouse AlexaFluor555 (1:1000, IgG/A-31570, Molecular Probes/Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA). Hoechst 33342 was used as a DNA counterstain at 0.5 μg/mL (B2261, Sigma-Aldrich, St. Louis, MO, USA).
6
Protein Expression Analysis by Western Blot
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Cytoplasmic protein and nuclear protein were extracted using a Nuclear and Cytoplasmic Extraction Reagents kit (78835, Thermo Fisher Scientific, USA). The protein concentration was measured by the BCA Assay Kit (23223, Thermo Scientific, USA). The protein was electrophoresed with 10% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, and subsequently transferred to the polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Specific primary antibodies (ab181602, ab32535, ab39012, ab41037, ab188570, ab186414, ab32042, ab32561, ab219413, ab76429, ab133462, ab32536, ab32511, Abcam, Cambridge, UK) (PA5-61136, Invitrogen, USA) were co-incubated with the membrane overnight at 4 °C. The membranes were rewarmed for 2 h, and then incubated with anti-rabbit IgG (ab97051, Abcam, Cambridge, UK) at 37 °C for 2 h. ECL Western blot kit (32209, Thermo Fisher Scientific, USA) was used to detect the bands. GAPDH and Lamin B were used as controls.
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