For most stainings, embryos were fixed with 3% PFA in 80 mM K+ Pipes, pH 6.8, containing 2 mM MgCl2 and 5 mM EDTA for 2 h (Werner and Mitchell, 2013 (link)). For AE1, acetylated tubulin, γ-tubulin, and CLAMP mAb staining, embryos were fixed in 100% ice-cold methanol followed by rehydration in EtOH. RPE cells were fixed with 3% PFA in 80 mM K+ Pipes, pH 6.8, containing 2 mM MgCl2 and 5 mM EDTA for 10 min or 100% ice-cold methanol for 20 min. RPE cells and embryos were blocked with 5% normal donkey serum in PBS with 0.1% Triton X-100 for 1 h. The following primary antibodies were used according to the manufacturer’s recommended dilutions: mouse anti–β-tubulin (7–10) and mouse anti-AE1 (IVF12; both from the Developmental Studies Hybridoma Bank), mouse anti-acetylated α-tubulin (T7451; Sigma-Aldrich), mouse anti–γ-tubulin (GTU-88; Sigma-Aldrich), rabbit anti–ZO-1 (61-7300; Invitrogen), and Cy-2–, Cy-3–, or Cy-5–conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.). Phalloidin 568 or 647 (Invitrogen) were used to visualize actin. Embryos were mounted between two coverslips using Fluoro-Gel (Electron Microscopy Sciences; Werner and Mitchell, 2013 (link)).
Mouse anti γ tubulin gtu 88
Manufactured by Merck Group
The Mouse anti-γ-tubulin GTU-88 is an antibody that recognizes the gamma-tubulin protein. Gamma-tubulin is a key component of the microtubule-organizing center (MTOC) and plays a crucial role in the nucleation and organization of microtubules. This antibody can be used to detect and localize gamma-tubulin in various cell types and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Fluoview upright confocal microscope, by Olympus (2 mentions)
Rabbit anti acetylated tubulin, by Cell Signaling Technology (2 mentions)
Fluoview fv10 asw software, by Olympus (2 mentions)
Ab201227, by Abcam (2 mentions)
Mouse anti γ adaptin, by BD (2 mentions)
Alexa fluor 640, by Jackson ImmunoResearch (2 mentions)
Rat anti mouse lamp2 gl2a7, by Abcam (2 mentions)
Alexa fluor 680, by Jackson ImmunoResearch (2 mentions)
Alexa fluor 790, by Jackson ImmunoResearch (2 mentions)
Mouse anti gfp clones 7 1 and 13, by Roche (2 mentions)
Alexa fluor 488, by Jackson ImmunoResearch (2 mentions)
Alexa fluor 594, by Jackson ImmunoResearch (2 mentions)
Rat anti ha 3f10, by Roche (2 mentions)
Ta99 mel 5, by American Type Culture Collection (2 mentions)
Mouse anti acetylated α tubulin, by Merck Group (1 mentions)
Phalloidin 568 or 647, by Thermo Fisher Scientific (1 mentions)
Rabbit anti zo 1, by Thermo Fisher Scientific (1 mentions)
Fluoro gel, by Electron Microscopy Sciences (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Axioskop 2 microscope, by Zeiss (1 mentions)
11 protocols using mouse anti γ tubulin gtu 88
1
Embryo and RPE Cell Immunostaining Protocol
2
Immunofluorescence Imaging of Zebrafish Embryos
3
Immunofluorescence Imaging of Zebrafish Embryos
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Zebrafish embryos were fixed in Dent’s fix (80% methanol, 20% DMSO), for at least 3 hr at room temperature or with fish fixative overnight at 4°C. Fixed embryos were stored in methanol at −20°C. Embryos were washed in a decreasing methanol:PBS gradient, followed by a PBS wash and blocking in PBDT (1% (w/v) BSA, 1% DMSO, 0.5% Triton X-100, PBS base) for 1 hr. Primary antibodies were added to PBDT and incubated with the embryos at 4°C overnight. Embryos were then washed in PBDT before incubating with fluorophore-conjugated secondary antibodies and DAPI (4′,6-diamidino-2-phenylindole; Invitrogen #D1306) for 3 hr at room temperature. The embryos were then stored in 70% glycerol, mounted, and imaged using an Olympus Fluoview Upright Confocal Microscope. Image acquisition and analysis was carried out using Olympus Fluoview FV10-ASW software. The primary antibodies used were: mouse anti-myosin heavy chain A4.1025 (Developmental Studies Hybridoma Bank, 1:20), rabbit anti-acetylated tubulin (Cell Signaling Technology #5335) and mouse anti-γ-tubulin GTU-88 (Sigma #T6557) (both 1:500). DAPI was used to label cell nuclei.
4
Immunostaining and Imaging of Drosophila Larval Brains
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Fixation and stainings of third instar larval brains was performed as previously described [53 (link)]. The MT regrowth assay in larval brains was performed as described in [56 (link)]. The following primary antibodies were used at a 1:500 dilution: Rabbit anti-Dlic (this study—raised against amino acids 1–293 of the Drosophila Dlic (CG1938) coding sequence; Nina Peel, personal communication), rat anti-Asl [73 (link)], rabbit anti-Asl (N-terminal and C-terminal) [37 ], guinea pig anti-Cnn [45 (link)], rabbit anti-Spd-2 [43 (link)], mouse anti- γ-tubulin (GTU88, Sigma), mouse anti-actin (Sigma-Aldrich), rabbit anti-PLP [53 (link)], rabbit anti-TACC [74 (link)], rabbit anti-Grip71WD [75 (link)], rabbit anti-Msps [76 ], rabbit anti-Aurora A [77 (link)], mouse monoclonal anti-α-tubulin (DM1α, Sigma-Aldrich) and rabbit anti-Histone H3 Phospho S10 (Upstate Biotechnology). Alexa488 anti-guinea pig and anti-mouse and Alexa568 anti-rabbit as secondary antibodies were used at a 1:1000 dilution (Molecular Probes, Life Technologies). Fixed preparations were examined using either a Zeiss LSM780 confocal microscope using a 63x/1.40 NA objective, or on a Zeiss Axioskop 2 microscope (Carl Zeiss, Ltd) with a CoolSNAP HQ camera (Photometrics), using a 63x/1.25 NA objective (Carl Zeiss, Ltd). Images were processed with Fiji [78 ] and adjusted to use the full range of pixel intensities.
5
Immunohistochemistry of Zebrafish Embryos
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Embryos were fixed for 2 h at room temperature in 4% paraformaldehyde in PBS (PFA). Embryos were stored in methanol, and then rehydrated in a gradient of PBS/methanol. Embryos were permeabilized in acetone, blocked in PBDT (PBS, 1% BSA, 1% DMSO and 1% Triton X-100) plus 5% sheep serum. Primary and secondary antibodies were incubated and washed in PBDT.
Primary antibodies used include mouse anti-acetylated-α-tubulin 6-11B-1 (1:500 for zebrafish embryos, Sigma, #T6793), mouse anti-γ-tubulin GTU-88 (1:500, Sigma, #T6557), rabbit anti-GFP (1:500, Abcam, #ab6556; Torrey Pines Biolabs, #TP401), rabbit anti-Arl13b (Duldulao et al., 2009 (link)) (1:200), DAPI (4′,6-diamidino-2-phenylindole; Molecular Probes, #D1306) was used to label cell nuclei. Alexa fluorophore-labeled anti-rabbit or anti-mouse secondary antibodies (Molecular Probes) were used to visualize staining.
Primary antibodies used include mouse anti-acetylated-α-tubulin 6-11B-1 (1:500 for zebrafish embryos, Sigma, #T6793), mouse anti-γ-tubulin GTU-88 (1:500, Sigma, #T6557), rabbit anti-GFP (1:500, Abcam, #ab6556; Torrey Pines Biolabs, #TP401), rabbit anti-Arl13b (Duldulao et al., 2009 (link)) (1:200), DAPI (4′,6-diamidino-2-phenylindole; Molecular Probes, #D1306) was used to label cell nuclei. Alexa fluorophore-labeled anti-rabbit or anti-mouse secondary antibodies (Molecular Probes) were used to visualize staining.
6
Immunofluorescence Imaging of AURKA and γ-Tubulin
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Cells were seeded at 2×104 onto glass coverslips and then fixed with cold 100% methanol (−20°C), permeabilized with 0.5% Triton X-100 in PBS and incubated in 2% BSA, 0.2% Triton X-100 in PBS (blocking buffer) for 1 h at room temperature. Cells were incubated overnight with primary antibodies (rabbit anti-phospho-AURKA Thr288, Cell Signaling Technology, 1:50; mouse anti-γ-tubulin GTU-88, Sigma-Aldrich, 1:1000) diluted in blocking buffer at 4°C, then washed three times with blocking buffer and incubated with secondary antibodies at 1:1000 dilution. Alexa Fluor 488 anti-mouse and Alexa Fluor 568 anti-rabbit (Thermo Fisher Scientific) were used as the secondary antibodies. DNA was stained with DAPI. Coverslips were mounted with Prolong Gold antifade reagent. Epifluorescence stacks were acquired using 500 nm z step with 2×2 bin using appropriate filter sets and a 40× NA 1.3 oil objective. The best in-focus images were selected and integrated intensities were measured using ImageJ (http://rsb.info.nih.gov/ij/ ; National Institutes of Health, Bethesda, MD).
7
Antibody Dilutions for Immunoblotting and IF
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The following antibodies were used for immunoblotting and IF at the indicated dilutions: rabbit anti-TRF1 1449 (1:2000), rabbit anti-BLM Ab2179 (1:500; Abcam), rabbit anti-TIN2 1447 (1:2000), rabbit anti-53BP1 NB100-304 (1:1000; Novus Biologicals), mouse anti-Myc 9E10 (1:1000; Cell Signaling), mouse anti-Flag M2 (1:1000; Sigma-Aldrich), mouse anti-γH2AX JBW301 (1:1000; Millipore), mouse anti-HA 12CA5 (1:1000; Roche), and mouse anti-γ-Tubulin GTU-88 (1:10,000; Sigma-Aldrich).
8
Immunofluorescence Microscopy Protocols
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mAbs and their sources included mouse anti-TYRP1 (TA99, also known as Mel-5; American Type Culture Collection) and mouse anti-Myc for dIFM (9E10; American Type Culture Collection), mouse anti-Pldn (clone 2G6; a generous gift from Esteban Dell’Angelica, University of California, Los Angeles, CA; Ghiani et al., 2010 (link)), mouse anti–AP-3δ (clone SA4; Developmental Studies Hybridoma Bank), rabbit anti-AP3M1 (ab201227; Abcam), mouse anti–γ-tubulin (GTU88; Sigma-Aldrich), mouse anti-VAMP7 (clone 158.2; Synaptic Systems), mouse anti-GFP (NB600-597, Novus Biologicals; or clones 7.1 and 13.1, Roche), and mouse anti-clathrin heavy chain (clone 23; BD Biosciences). Polyclonal antibodies included rabbit anti-STX13 (Prekeris et al., 1998 (link)), as previously described (Setty et al., 2007 (link)); rabbit anti-VAMP7 TG50, a kind gift from Thierry Galli (Verraes et al., 2018 (link)); rabbit αPep7h-msm to the cytoplasmic domain of TYR (Berson et al., 2000 (link)); rabbit anti-dysbindin (Starcevic and Dell’Angelica, 2004 (link)), a kind gift from Esteban Dell’Angelica; and sheep anti-TGN46 (Serotec). Species-specific secondary antibodies from donkey and conjugated to Alexa Fluor 488, Cy3, Alexa Fluor 594, and Alexa Fluor 647 were used for dIFM or to IRDye-790CW, IRDye-680LT, or HRP for immunoblotting (Jackson ImmunoResearch Laboratories, Inc.).
9
Characterization of Intracellular Trafficking Proteins
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Primary monoclonal antibodies used and their sources (indicated in parentheses) were: mouse anti-TYRP1 (TA99/Mel-5; American Type Culture Collection); mouse anti-γ-Tubulin (GTU-88; Sigma-Aldrich); mouse anti-γ-adaptin (610385; BD); mouse anti-GFP (clones 7.1 and 13.1; Roche); rabbit anti-AP3M1 (ab201227; Abcam); rat anti–mouse LAMP2 (GL2A7; Abcam); rat anti-mouse TfR (CD71; BD 553264); and rat anti-HA (3F10; Roche 11867423001). Primary polyclonal antibodies used and their sources were: rabbit anti-STX13 (a kind gift of Rytis Prekeris, University of Colorado, Denver, CO; Prekeris et al., 1998 (link)); rabbit anti-pallidin (a kind gift of Juan Bonifacino, National Institute of Child Health and Human Development, Bethesda, MD; Moriyama and Bonifacino, 2002 (link)); rabbit anti-PI4KIIIβ (13247-1-AP; Proteintech); rabbit anti-PI4KIIα and anti-PI4KIIβ (kind gifts of Pietro De Camilli, Yale University, New Haven, CT; Guo et al., 2003 (link)); and rabbit anti-GFP (PABG1; Chromotek); Species- and/or mouse isotype–specific secondary antibodies from donkey or goat conjugated to Alexa Fluor 488, Alexa Fluor 594, or Alexa Fluor 640 used for IFM or to Alexa Fluor 680 or Alexa Fluor 790 for immunoblots were obtained from Jackson ImmunoResearch Laboratories.
10
Microtubule Dynamics Analysis in Insect Cells
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Nocodazole, Latrunculin A, Dimethyl sulfoxide (DMSO), and Shields and Sang M3 Insect Medium were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nocodazole was dissolved at 1 mg/mL in DMSO and stored frozen at −20 °C. We used the following antibodies: rat anti-α-tubulin-YL1/2 (1:50; ab6160 Abcam), mouse anti-acetylated tubulin (1:100; T7451 Sigma-Aldrich), mouse anti-α-tubulin (1:500; T5168 Sigma-Aldrich), mouse anti-γ-tubulin GTU88 (1:100; T6557 Sigma-Aldrich), mouse anti-polyglutamylated tubulin GT335 (1:200; T9822 Sigma-Aldrich), rabbit anti-centrosomin (Cnn, 1:400; [23 (link)]), rabbit anti-Spd-2 (1:500; [24 (link)]), and rabbit anti-pavarotti (Pav-KLP, 1:400; [25 (link)]). The secondary antibodies used (1:800), Alexa Fluor 488, 555, and 647 anti-mouse, anti-rabbit, anti-rat, and anti-chicken, were obtained from Invitrogen. Hoechst 33,258 was obtained by Sigma-Aldrich.
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