Brain sections were co-immunostained with a goat (sc-7396, 1:1000, Santa Cruz Biotechnology) or rabbit (T5192, 1:500, Sigma-Aldrich) anti-γ-tubulin pAb, mouse anti-ZO1 mAb (33–9100, 1:200, Life Technologies) and chick (GFP-1020, 1:1000, Aves Labs) or rabbit (598, 1:1000, MBL) anti-GFP pAb, along with DAPI nuclear staining (Thermo Fisher Scientific). Confocal images were captured by using an FV1000 microscope (Olympus) with a × 40 or × 60 objective lens and 0.5-μm interval-Z scanning. Images of mitotic spindles in sections were 3D reconstructed by ImageJ software, and spindle orientation was measured by using the R package39 (link).
T5192
Manufactured by Merck Group
Sourced in United States
The T5192 is a laboratory equipment product from Merck Group. It is a precise and reliable tool designed for general laboratory applications. The core function of the T5192 is to perform specific laboratory tasks with consistent and accurate results.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (3 mentions)
Pierce ecl, by Thermo Fisher Scientific (3 mentions)
T9026, by Merck Group (2 mentions)
Ab10294, by Abcam (2 mentions)
Sc 32308, by Santa Cruz Biotechnology (2 mentions)
Sc398326, by Santa Cruz Biotechnology (2 mentions)
A1978, by Merck Group (2 mentions)
Anti cortactin mouse, by Santa Cruz Biotechnology (2 mentions)
Sc 55579, by Santa Cruz Biotechnology (2 mentions)
Universal immune peroxidase anti mouse, by Nichirei Biosciences (2 mentions)
Phalloidin ifluor 647 reagent, by Abcam (2 mentions)
T5326, by Merck Group (2 mentions)
T5168, by Merck Group (2 mentions)
A11008, by Thermo Fisher Scientific (2 mentions)
Phalloidin ifluor 488 reagent, by Abcam (2 mentions)
Sc 7396, by Santa Cruz Biotechnology (1 mentions)
Dapi nuclear staining, by Thermo Fisher Scientific (1 mentions)
Fv1000 microscope, by Olympus (1 mentions)
Gfp 1020, by Aves Labs (1 mentions)
Ab75783, by Abcam (1 mentions)
13 protocols using t5192
1
Visualizing Mitotic Spindle Orientation
2
Immunofluorescence Imaging of Mitotic Spindle
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HeLa cells were cultured on ethanol-sterilized coverslips in 12-well plates and were synchronized or monastrol-treated before fixation with ice-cold methanol at −20 °C for 10 min, followed by rehydration with 1 × PBS at room temperature for 10 min. The fixed cells were permeabilized with 0.3% Triton X-100 in 1 × PBS for 15 min and blocked with 2% bovine serum albumin for 45 min at room temperature. Mouse anti-α-tubulin 1:2,000 (T9026, Sigma), rabbit anti-γ-tubulin 1:200 (T5192, Sigma), human anticentromere antibody 1:2,000 (HSM0101, ImmunoVision), rabbit anti-Kid 1:200 (ab75783, Abcam) and rabbit anti-NuSAP 1:200 (ab93779, Abcam) were utilized to stain the respective proteins in cells. Alexa Fluor dye-conjugated goat anti-mouse, goat anti-rabbit or goat anti-human IgG (Invitrogen) were used as the secondary antibodies. DNA was stained with Hoechst 33342 (Invitrogen). Cells were mounted in FluorSave reagent (Calbiochem). The images were processed using either the Volocity software (PerkinElmer) or Image J (National Institutes of Health, Bethesda, MD), as required.
3
Immunofluorescence Microscopy Protocol
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For immunofluorescence microscopy, the following antibodies were used: chicken anti-myc (1:300, A-21281; Molecular Probes, Eugene, OR), rabbit anti-SSX2IP (1:150, HPA027306; Sigma-Aldrich, Gillingham, United Kingdom), rabbit anti-PCM1 (1:300, sc67204; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti–γ-tubulin (1:250, T5192; Sigma-Aldrich), mouse anti–α-tubulin (1:250, T9026; Sigma-Aldrich), rabbit anti–centrin-2 (1:400, sc27793R; Santa Cruz Biotechnology), mouse anti-Plk4 (1:200, H00010733-B01; Abnova, Taipei, Taiwan), rabbit anti-Cep152 (1:400, A302-480A; Bethyl Laboratories, Montgomery, TX), rabbit anti-hSAS-6 (1:100, sc98506; Santa Cruz Biotechnology), mouse anti-centrobin (1:100, H00116840-B01P; Abnova), rabbit anti-Cep164 (1:200, 45330002; Novus Biologicals, Littleton, CO), mouse anti–C-Nap1 (1:100, 611374; BD Biosciences, San Jose, CA), and rabbit anti-CP110 (1:100, ab99337; Abcam, Cambridge, MA). Secondary antibodies were Alexa Fluor 488–coupled anti-rabbit, Alexa Fluor 594–coupled anti-rabbit, Alexa Fluor 594–coupled anti-mouse, Alexa Fluor 488–coupled anti-mouse, Alexa Fluor 488–coupled anti-chicken, and Cy3-coupled anti-mouse antibodies (all used at 1:1500). For immunoblotting, rabbit anti-SSX2IP (1:1000) and mouse anti–α-tubulin (1:5000) antibodies were used.
4
Characterization of CLPTM1L Protein Localization
5
Antibodies Used for Cell Organelle Analysis
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The following antibodies were used in this study: mouse anti–Ac-tubulin (1:400; T6793, Sigma), rabbit anti-Arl13b (1:100; 17711–1-AP, Proteintech), rabbit anti–γ-tubulin (1:200; T5192, Sigma), rabbit anti-CP110 (1:200; 12780–1-AP, Proteintech), rabbit anti-AurA (1:1,000; 4718S, CST), rabbit anti-HDAC6 (1:1,000; 7612, CST), mouse anti-calregulin (1:400; sc-11398, Santa Cruz), mouse anti-TOM20 (1:400; sc-17764, Santa Cruz), and rabbit anti-Rab7 (Western blot 1:1,000, immunofluorescence 1:200; ab137029, Abcam).
6
Immunofluorescence Staining of RPE1 Cells
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RPE1 cells were fixed in −20°C methanol (Thermo Fisher Scientific) and 1% paraformaldehyde (Electron Microscopy Sciences). Cells were then washed in 1× TBS and blocked in antibody dilution buffer (Abdil; TBS pH 7.4, 1% BSA, 0.1% Triton X-100, and 0.1% sodium azide) containing 20% goat serum. Cells were incubated with the following primary antibodies for 1 h at room temperature in Abdil: mouse anti-human lamin A/C (1:200 in cells or 1:1,000 for tissue sections, MAB3211; Millipore), rabbit anti-lamin A/C (1:200 in cells or 1:1,000 for tissue sections, ab26300; Abcam), rabbit anti-lamin B1 (1:200, ab16048; Abcam), rabbit anti-γH2AX (1:200, 9718S; Cell Signaling Technologies), rabbit anti-mCherry (1:200, ab167453; Abcam), mouse anti-γ-tubulin (1:200, T5192; Sigma-Aldrich), and rabbit anti-ezrin (1:1,000, 3145; Cell Signaling Technologies). Cells were incubated overnight at 4°C with human ACA (1:200, 15235; Antibodies Inc.). Cells were incubated for 1 h at room temperature, in the dark, with goat secondary antibodies against mouse, rabbit, or human IgG conjugated to Alexa Fluor 488, 594, or 647 (Molecular Probes by Life Technologies). Coverslips were mounted on glass slides with ProLong Gold Antifade Reagent plus DAPI (Thermo Fisher Scientific).
7
Immunoblotting of hnRNPC in cisplatin-treated cells
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T6a cells were harvested 24 (siRNA-transfected) or 48 (pcDNA vectors-transfected) hours after cisplatin treatment and re-plating. H2009 cells were harvested 48 hours after siRNA of pcDNA transfection. Cells were then lysed in RIPA buffer and subjected to standard SDS/PAGE electrophoresis and transferred to nitrocellulose membranes. The membranes were immunoblotted with antibodies against hnRNPC (T6a - ab10294, Abcam Inc.; H2009 - sc-32308, Santa Cruz) and γ-tubulin (T5192, Sigma-Aldrich) following manufacturer’s instructions.
8
Immunocytochemistry of Cytoskeletal Proteins
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HeLa cells were fixed in methanol for 5 min at −20°C and permeabilized in 0.5% Triton X-100. The cells were then incubated overnight at 4°C with antibody against β-tubulin (1 µg/ml; mouse, E1C601-2; Eno Gene) or γ-tubulin (1:2,000; rabbit, T5192; Sigma-Aldrich) in PBS containing 0.1% BSA and 0.1% Triton X-100. Cells were washed three times with PBS, followed by incubation for 1 h at room temperature in the dark with Alexa Fluor 594 goat anti–rabbit and Alexa Fluor 488 goat anti–mouse antibodies (both at a 1:3,000 dilution; Invitrogen) in PBS containing 0.1% BSA and 0.1% Triton X-100. The cells were further incubated with 0.1 µg/ml Hoechst 33258 in PBS for 1 min, followed by extensive washing with PBS, and then photographed with a charge-coupled device camera (Axio Observer Z1; ZEISS).
9
Antibody Staining Protocol for Cellular Analysis
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Anti-ORP3 rabbit antibody (1:2000 for WB and 1:400 for IF, A304-557A, Bethyl Laboratories, Texas, USA). Anti-ORP3 mouse antibody (1:400 for IHC and 1:2000 for WB, sc398326, Santa Cruz, California, USA). Anti-Cortactin mouse antibody (1:500 for IF, sc-55579, Santa Cruz, California, USA). Anti-α-tubulin mouse antibody (1:2000 for WB, T5168, Sigma-Aldrich, St. Louis, USA). Anti-γ-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5326, Sigma-Aldrich, St. Louis, USA). Anti-γ-tubulin rabbit antibody (1:500 for IF, T5192, Sigma-Aldrich, St. Louis, USA). Anti-β-actin mouse antibody (1:10,000 for WB, A1978, Sigma-Aldrich, St. Louis, USA). Secondary antibodies, HRP anti-Mouse antibody (1:5000 for WB, 7076S, Cell Signaling Technology, Danvers, USA) and HRP anti-Rabbit (1:2000 for WB, 7074S, Cell Signaling Technology, Danvers, USA). One drop of Universal immune-peroxidase anti-Mouse (Nichirei Bioscience, Tokyo, JP) per piece of tissue was used in IHC. Phalloidin-iFluor 488 Reagent (1:1000 for IF, ab176753, Abcam, Cambridge, UK). Phalloidin-iFluor647 reagent (1: 1000 for IF, ab176759, Abcam, Cambridge, UK). Anti-Rabbit conjugated with Alexa Fluor488 antibody (1:1000 for IF, A11008, ThermoFisher Scientific, Waltham, USA), and anti-Mouse conjugated with Cy5 antibody (1:200 for IF, 115–605-006, ThermoFisher Scientific, Waltham, USA).
10
Immunoblotting of hnRNPC in cisplatin-treated cells
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