Immunofluorescence was performed as previously described59 (link). Antibodies were purchased from Sigma (α-tubulin, γ-tubulin, centrin and Flag), Abcam (γH2AX) and Invitrogen (Alexa Fluor 568-conjugated IgG secondary antibody). Of note, cells were incubated at 4 °C for 1 h before permeabilization and fixation for centriole staining9 (link).
γ tubulin
Manufactured by Abcam
Sourced in United States
γ-tubulin is a protein that is a component of the microtubule organizing center (MTOC) in eukaryotic cells. It plays a key role in the nucleation and organization of microtubules during cell division and other cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Acetylated α tubulin, by Merck Group (6 mentions)
Krt17, by Abcam (6 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (6 mentions)
Krt14, by Fortrea (6 mentions)
α tubulin, by Abcam (4 mentions)
Bromodeoxyuridine (brdu), by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (4 mentions)
Ds qi1mc camera, by Nikon (4 mentions)
Rpgrip1l, by OriGene (2 mentions)
Phospho histone h3, by Cell Signaling Technology (2 mentions)
α tubulin, by Merck Group (2 mentions)
Mounting medium with dapi, by Vector Laboratories (2 mentions)
Ds qi1mc, by Nikon (2 mentions)
Gfp yfp, by Aves Labs (2 mentions)
Gorab, by Proteintech (2 mentions)
Lectin helix pomatiaagglutinin hpa, by Thermo Fisher Scientific (2 mentions)
Golgin 97, by Thermo Fisher Scientific (2 mentions)
Deadend fluorometric tunel system, by Promega (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Centrin, by Abcam (1 mentions)
22 protocols using γ tubulin
1
Immunofluorescent Staining of Centrioles
2
Immunofluorescence and TUNEL Staining Protocol
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Immunofluorescence and TUNEL staining were performed as described previously (Dai et al., 2013 (link)). The following primary antibodies were used: RPGRIP1L, 1:100 (Origene, Rockville, MD); p63, 1:100 (Santa Cruz Biotechnology, Santa Cruz, CA); KRT14, 1:1,000 (Covance, Princeton, NJ); KRT1, 1:500 (Roop et al., 1987 (link)); KRT17, 1:1,000 (Abcam, Cambridge, MA); LOR, 1:100 (Covance); NGFR, 1:100 (Promega); LEF1, 1:100 (Cell Signaling, Danvers, MA); BrdU, 1:50 (Life Technologies); acetylated α-tubulin, 1:1,000 (Sigma, St. Louis, MO); γ-tubulin, 1:1,000 (Abcam); ARL13B, 1:1,000 (#73-287, NeuroMab, Davis, CA); phospho-histone H3, 1:100 (Cell Signaling). AlexaFluor-conjugated secondary antibodies (1:250) were from Life Technologies. Sections were sealed in mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Images were acquired by Nikon 80i fitted with Nikon DS-Qi1Mc camera and processed with Photoshop 5.5 CS.
3
Immunofluorescence of Ventricular Walls
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Medial and lateral ventricular walls from P10 mice were isolated in cold PBS and sectioned using a Leica VT1000S vibratome (Leica, Wetzlar, Germany). Tissue was fixed in 4% PFA in PBS O/N at 4°C, followed by blocking with 2.5% BSA, 0.2% Triton X-100 in PBS for 4 hours at RT. Primary antibodies used: N-cadherin (rabbit, 1:50; sc-7939, Santa Cruz Biotechnology, Santa Cruz, CA), γ-tubulin (mouse 1:250; ab11316, Abcam, Cambridge, MA). Samples were incubated with primary antibody for 24–48 hours at 4°C. Secondary antibody incubation was performed at 4°C for 48 hours using Alexa 488 goat anti-mouse (1:250, Thermo-Fisher, Waltham, MA) and Alexa 647 donkey anti-rabbit (1:250, Jackson ImmunoResearch, West Grove, PA). Tissues were placed on glass slides, mounted and imaged using an Andor WD Spinning Disk confocal microscope system equipped with an Andor Neo sCMOS camera (Andor, Belfast, UK).
4
Immunofluorescence Staining of Cell Markers
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Primary antibodies to the following proteins were used in this study: calreticulin (C4606; Sigma-Aldrich), Tuba (a gift from Frank Gertler, MIT; and B01P, Abnova), centrin2 (a gift from Jeffery L. Salisbury, Mayo Clinic; and Clone 20H5, Millipore), FGD1 (HPA 000911; Sigma-Aldrich), myc (Clone 9E10; Cal Biochem), GM130 (Clone 35; BD Biosciences; and G7295, Sigma-Aldrich), giantin (a gift from Vivek Malhotra, Center for Genomic Regulation, Barcelona, Spain), kendrin (a gift from Mikiko Takahashi, Teikyo Heisei University), α-tubulin (T5168; Sigma-Aldrich), γ-tubulin (ab11310; Abcam), Cdc42 (Clone 44; BD Biosciences), and vesicular stomatitis virus G-protein (VSVG; BWG85; a gift from Victor Hsu, Harvard Medical School). Secondary antibodies for immunofluorescence were from Theromo Fisher or Biotium, and near-infrared antibodies for Western blots were from LI-COR (Lincoln, NE). For immunofluorescence, cells were fixed in either 100% ice-cold methanol (JT Baker) or 4% formaldehyde (Ted Pella), blocked and permeabilized with 2% blocking buffer (2% fetal bovine serum [FBS], 0.01% Triton X-100, and 1× phosphate-buffered saline), stained with primary antibodies for 1 h at room temperature, and stained with secondary antibodies for 1 h. Coverslips were mounted with ProLong Gold (Thermo Fisher), and imaging dishes were filled with Ibidi Mounting Medium (Ibidi).
5
Immunofluorescence Analysis of Cell Adhesion and Cytoskeleton
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The samples on the nanofabricated coverslip were fixed with ice-cold 4% paraformaldehyde for 20 min, washed two times with phosphate-buffered saline (PBS), and permeablized with 0.1% Triton X-100 in PBS for 5 min. After washing with PBS, cultures were blocked with 10% goat serum for 1 h, and then incubated with primary antibodies against Vinculin (1:200, Sigma-Aldrich), E-cadherin (1:200, Cell Signaling, 3195), phospho-Myosin Light Chain (1:200, Cell Signaling, 3674), γ-tubulin (1:100, Abcam, ab11321), YAP (1:100, Cell Signaling, 4912 and Santa Cruz, sc-15407), active β-catenin (1:100, Millipore, 05-665), Vimentin (1:100, Abcam), Slug (1:100, Cell Signaling, 9585), Twist (1:100, Santa Cruz, sc-15393), Vimentin (1:100, Abcam, ab24525), and WT1 (1:100, Santa Cruz, sc-192) for 3 h at room temperature. After washing with secondary antibodies and Alexa Fluor 594 conjugated phalloidin (1:40, Molecular Probes) and Hoescht (Invitrogen), cultures were incubated for 1 h at room temperature. The slides were mounted with an anti-fade reagent (SlowFade gold, Invitrogen) and taken by an inverted microscope (Zeiss Axiovert 200 M) with an X40 oil immersion objective (Zeiss, 1.6 NA).
6
Immunoblotting, Immunofluorescence, and FACS Protocols
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Immunoblotting, immunofluorescence and FACS assays were performed as previously described (14 (link)). Antibodies that were employed to perform these studies were the following: p53 (1:500; mouse monoclonal; cat. no. PIMA512557; Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA), p21 (1:500; mouse monoclonal; cat. no. AHZ0422; Thermo Fisher Scientific), centrin 20H5 (1:1,000; mouse monoclonal; Mayo Clinic, Rochester, MN, USA), pericentrin (1:500; rabbit polyclonal; cat. no. ab4448l) and p-retinoblastoma (1:400; rabbit polyclonal; cat. no. ab47763; both from Abcam, Cambridge, MA, USA), γ-tubulin (1:4,000; mouse monoclonal; cat. no. MA1-850; Thermo Fisher Scientific), Aurora-A (1:500; mouse monoclonal; cat. no. ab13824; Abcam), p-Aurora-A (1:400; rabbit monoclonal; cat. no. 3079S; Cell Signaling Technology, Inc., Danvers, MA, USA), p-retinoblastoma (1:500; mouse monoclonal; cat. no. R6878-1ML) and β-actin (1:5,000; mouse monoclonal, cat. no. A2228-100UL; both from Sigma-Aldrich, St. Louis, MO, USA). Results were derived from three independent experiments with comparable outcomes.
7
Lateral Ventricular Wall Immunostaining
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Lateral ventricular walls were dissected and immediately immersed in PBS as described previously.8 (link) Whole-mount preparations were fixed in 4% paraformaldehyde and 0.1% Triton X-100 for 12 min, washed three times in PBS-0.1% Triton X-100, blocked in PBS supplemented with 0.5% Triton X-100 and 3% BSA, and incubated with β-Catenin antibody (Cell Signaling, Danvers, MA, USA, Cat no. 9581, 1/500), ZO-1 (Invitrogen, Brussels, Belgium, Cat no. 61-7300, 1/200), γ-Tubulin (Abcam, Cat no. ab11317, 1/500) and GFAP (Millipore, Cat no. AB5804, 1/1000).
8
Immunofluorescence Labeling Protocols for Cell Analysis
9
Immunofluorescence Microscopy of ASK1 and EB1
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Primary antibodies directed against ASK1 (Abcam), EB1 (BD Biosciences), α-tubulin (Abcam), γ-tubulin (Abcam), phosphoserine (Cell Signaling Technology), phosphothreonine (Cell Signaling Technology), and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz), rhodamine- or fluorescein-conjugated secondary antibodies (Jackson ImmunoResearch) were obtained from the indicated sources. The pT206-EB1 customized antibody was obtained from GL. DAPI, and fluorescein-conjugated phalloidin were purchased from Sigma-Aldrich. The control siRNA (5′-CGUACGCGGAAUACUUCGA-3′) and ASK1 siRNAs (#1: 5′-GCACUCCU-UCAUCGAGCU-3′; #2: 5′-GGUAUACAUGAGUGGAAUU-3′) were synthesized by Ribo Bio. Mammalian expression plasmids for GFP-EB1 were constructed by PCR using the pEGFP-N1 vector as described previously [31 (link)].
10
Immunofluorescence and TUNEL Staining Protocol
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