For the staining of primary cilia, the cells were washed with cold phosphate-buffered saline (PBS) and fixed with 4% (w/v) paraformaldehyde, which was dissolved in PBS containing 0.1% (v/v) Triton X-100. Subsequently, cells were blocked with PBS containing 1% bovine serum albumin (BSA) and incubated overnight at 4 °C with primary antibodies against acetylated α-tubulin (1:1000; T7451, Sigma-Aldrich), γ-tubulin (1:1000; T5326, Sigma-Aldrich), and ARL13B (1:1000; 17711-1-AP, Proteintech) in 1% BSA. After washing, the cells were incubated with Alexa Fluor 488- or 555-conjugated secondary antibodies at room temperature (RT) for 1 h. Before mounting, the cells were treated with Hoechst 33,342 dye (1:10000, H3570, Thermo Fisher Scientific) for nuclear staining. Cilia were observed using a fluorescence microscope. Cilia were counted in approximately 200 cells for each experimental condition (n = 3). The ciliated cell percentage was calculated as follows: (total number of cilia/total number of nuclei in each image) × 100. Cilia lengths were measured using the free-hand line selection tool of Cell Sense Standards software (Olympus Europa Holding GmbH, Hamburg, Germany), and the average cilium length was calculated.
T5326
Manufactured by Merck Group
Sourced in United States
The T5326 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use, but a detailed product description is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (8 mentions)
T6199, by Merck Group (3 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (3 mentions)
P0067, by Merck Group (3 mentions)
Odyssey blocking buffer, by LI COR (3 mentions)
T5168, by Merck Group (3 mentions)
A412 1, by Fortis Life Sciences (2 mentions)
Ab52866, by Abcam (2 mentions)
Fluorochrome conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Nb600 506, by Novus Biologicals (2 mentions)
Immobilon fl membrane, by Merck Group (2 mentions)
Sc 6216, by Santa Cruz Biotechnology (2 mentions)
Phosstop, by Roche (2 mentions)
Supported nitrocellulose membrane, by Bio-Rad (2 mentions)
Ab290, by Abcam (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)
39 protocols using t5326
1
Immunofluorescent Cilia Visualization
2
Quantifying KIF18A Depletion in Mitosis
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After cells had been seeded onto 12 mm glass coverslips in 24-well plates for 24 hours endogenous KIF18A was depleted as described above and GFP-KIF18A was induced with 2 μg/mL doxycycline. 24 hours after knockdown and induction cells were fixed in −20°C methanol (Fisher Scientific, A412-1) for 3 minutes on ice and stained for rabbit KIF18A 1:100 (Bethyl, A301-080A), guinea Pig CENP-C 1:250 (MBL, PD030), and mouse gamma-tubulin 1:500 (Sigma Aldrich, T5326). All primary antibodies were incubated for 1 hour at room temperature, shaking. Twenty random mitotic cells were imaged for each condition per replicate, and expression level was quantified in ImageJ/Fiji by drawing an ROI around the mitotic spindle and measuring the KIF18A signal within that ROI. Background signal was determined by moving the same ROI to an area with no cells and measuring the GFP signal in that space. The mean background signal for each image was subsequently subtracted from the mean spindle KIF18A signal. All values were then normalized to the mean control background subtracted KIF18A intensity. Median and 95% confidence interval are reported for three individual biological replicates.
3
Analysis of γH2AX Foci and Centrosomal Localization
4
Centrosome Quantification in Serous Carcinoma
5
Seliciclib, a CDK2 Inhibitor, in Lung Cancer
6
Comprehensive Immunostaining Protocol
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Immunostaining was performed with the following primary antibodies: Ki67 1:500 (rabbit, NCL-ki67p; Novacastra, Wetzlar, Germany); mouse anti γ-tubulin (1:400, T5326 Sigma), rabbit anti γ-tubulin (1:400, T5192 Sigma), rabbit anti Arl13b (1:300, 17711-1-AP Proteintech), mouse anti α-tubulin (1:300, T5168 Sigma), mouse anti Arl13b (1:300, 75287, Antibodies Inc.), rabbit anti TBR1 (1:200, ab31940 Abcam), Monoclonal Anti-Acetylated Tubulin antibody produced in mouse clone 6-11B-1(Sigma Aldrich), rat anti TBR2 488 (1:200, 53-4875-80 eBioscience), rabbit anti phospho-histone3 (1:300, ab47297 Abcam), mouse anti PAX6 (1:200, DSBH), Cep55 (1:500; sc-374051Santa Cruz biotechnology), Pericentrin (1:1000; Covance, PRB-432C), β-Catenin (1:1000; Cell Signaling Technology, 9582), Cleaved Caspase-3 (1:500; 9664 Cell Signaling Technology), Tuj1 (TU20) (1:200, 4466s Cell Signaling Technology), GFP (1:500, AB290 Abcam), DAPI was used for the nuclear staining (D9564; MilliporeSigma). ApopTag staining was performed with an ApopTag peroxidase in situ apoptosis detection kit (S7100; MilliporeSigma, Billerica, MA, USA).
7
Quantifying Autophagy in HeLa Cells
8
Analyzing MYC Protein Stability in HEK293T Cells
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Protein stability assays were performed according to established methods54 (link). In brief, HEK293T cells were transfected using Xtreme-Gene (Roche 6366546001) following the manufacturer's instructions. After transfection, cells were incubated for 48 h in DMEM media supplemented with 10% FBS, then were treated with 100 μg/ml cycloheximide (2112, Cell Signaling) in DMEM media with 10% FBS. At the indicated times. 100 μl of 2X SDS-PAGE sample buffer was added and the cells were scraped from the wells, boiled for 5 min, then cell lysates were stored at −80 °C. After all lysates were collected, each sample was loaded onto a 10% SDS-PAGE gel and then analysed by western blotting with rabbit anti-MYC antibody (5605, Cell Signaling, 1:1,000) to monitor MYC protein level. Protein expression was quantified from the western blot using GelQuant software. For analysis, MYC levels were normalized to tubulin protein levels (mouse, T-5326, Sigma, 1:2,000). Assays were performed three times.
9
Centrosome Labeling for Microscopy
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Cells were seeded and fixed as for smFISH. Cells were permeabilized with 0.1% Triton-X100 in PBS for 10 min at room temperature and washed twice with PBS. For centrosome labeling, coverslips were incubated for 1 h at room temperature with a monoclonal anti-γ-tubulin antibody produced in mouse (Sigma-Aldrich, T5326), diluted 1/1000 in PBS. Coverslips were washed twice with PBS, 5 min each time, and incubated with either a FITC (Jackson ImmunoResearch 115-095-062) or Cy5 (Jackson ImmunoResearch 115-176-003) labeled anti-mouse secondary antibody diluted 1/100 in PBS. After 1 h of incubation at RT, coverslips were washed twice with PBS, 5 min each. Coverslips were mounted using Vectashield containing DAPI (Vector Laboratories, Inc.).
10
Centrosome Quantification in Serous Carcinoma
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Two-color immunofluorescence assay was used to measure the centrosome number per interphase cell in serous tubal intraepithelial carcinoma and high-grade serous carcinoma as previously described34 (link). Briefly, 5 μm sections from each tissue block was de-paraffinized in xylene then rehydrated through graded alcohols. Antigen retrieval was performed in Trilogy solution for 10 minutes. The slides were then incubated at room temperature for two hours with primary mouse-monoclonal γ-tubulin (T-5326, Sigma-Aldrich, dilution 1:200) and rabbit-monoclonal α-tubulin (ab52866, Abcam, 1:200). Signal detection was performed by incubating the slides with fluorochrome-conjugated secondary antibodies (from Jackson ImmunoResearch Laboratories) goat anti-mouse Rhodamine-Rd antibody (1:200), and goat anti-rabbit FITC (1:400) at room temperature for one hour. Three TBST washes were carried out before tissue sections were counterstained with DAPI and mounted.
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