Cells were fixed with 4% paraformaldehyde in PBS (Sigma-Aldrich) for 15 minutes and permeabilized with 0.5% Triton X-100 in PBS for 15 minutes. Slides were blocked with IB-solution for two hours and incubated either with rabbit polyclonal anti-Ccdc181 (1:100, Sigma-Aldrich) and mouse monoclonal anti-α-Tubulin (1:500, Sigma-Aldrich) or mouse monoclonal anti-acetylated α-Tubulin (1:2,000, Sigma-Aldrich), or with rabbit polyclonal anti-Ccdc181 (1:100, Bioss Antibodies) and anti-Hook1 (1:100, Bios Antibodies) directly coupled to Alexa488 or Alexa555, respectively, over night at 4°C. The next day, slides incubated with unlabeled primary antibodies were incubated with goat anti-mouse Alexa488- and goat anti-rabbit Alexa568-coupled antibodies (1:500, Invitrogen) for 90 minutes. Additionally, to visualize the acrosome of isolated germ cells all slides were incubated with Alexa647-conjugated PNA (1:100, Invitrogen) in PBS for 30 minutes. All slides were mounted using ProLong Diamond Antifade with DAPI (Invitrogen) and examined using the Zeiss LSM780 laser scanning microscope.
Mouse monoclonal anti acetylated α tubulin
Manufactured by Merck Group
Sourced in Germany, United States
Mouse monoclonal anti-acetylated α-tubulin is a laboratory reagent used to detect and visualize acetylated α-tubulin, a post-translational modification of the cytoskeletal protein α-tubulin.
Automatically generated - may contain errors
Lab products found in correlation
Mouse monoclonal anti α tubulin, by Merck Group (2 mentions)
Mouse monoclonal anti β catenin, by BD (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Lsm780 laser scanning microscope, by Zeiss (1 mentions)
Anti ccdc181, by Merck Group (1 mentions)
Prolong diamond antifade with dapi, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Axio observer lsm710 confocal microscope, by Zeiss (1 mentions)
Rat monoclonal anti cd11b, by Bio-Rad (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Alexa fluor 568 conjugated goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti vimentin, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi d9542, by Merck Group (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated donkey anti mouse, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 chicken anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti actin, by Merck Group (1 mentions)
Alexa fluor 546 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
8 protocols using mouse monoclonal anti acetylated α tubulin
1
Immunofluorescence Staining of Germ Cells
2
Immunofluorescence Labeling of Cellular Structures
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The cells were fixed with 4 % PFA, and unspecific binding was prevented by incubating cells in a 3 % BSA and 0.5 % Triton X-100 solution (all from Sigma-Aldrich) for 30 min at RT. The cells were incubated with primary antibodies prepared in 0.3 % BSA and 0.1 % Triton X-100, kept overnight at 4 °C, washed with PBS, and finally incubated for 1 h at RT with the corresponding secondary antibody. Antibodies used were as follows: rat monoclonal anti-CD11b (1:600; AbD Serotec), mouse monoclonal anti-acetylated α-tubulin (1:100; Sigma-Aldrich), goat polyclonal anti-Nox1 (1:250; Santa Cruz Biotechnology, Heidelberg, Germany), Alexa Fluor 594 goat anti-rat, Alexa Fluor 488 goat anti-rat, Alexa Fluor 488 donkey anti-goat, and Alexa Fluor 594 rabbit anti-mouse (all 1:200 in PBS, from Life Technologies Ltd). Membrane ruffling was observed using phalloidin, a marker for filamentous actin. The cells were incubated for 2 h with Alexa Fluor 594 conjugated to phalloidin (1:100; Life Technologies Ltd) in PBS, at RT. For nuclear labeling, cell preparations were stained with Hoechst 33342 (2 μg/ml, Life Technologies Ltd) in PBS, for 5 min at RT and mounted in Dako fluorescent medium. Fluorescent images were acquired using an Axio Observer LSM710 confocal microscope (Zeiss) under a 63× oil objective.
3
Immunofluorescence Staining of Cytoskeletal Markers
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The following commercial antibodies and reagents were used: mouse monoclonal anti–α-tubulin (T9026); mouse monoclonal anti-vimentin (V6630); mouse monoclonal anti-acetylated α-tubulin (T6793); tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin (P1951); 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, D9542) (Sigma-Aldrich, St. Louis, MO, USA); mouse monoclonal anti–β-catenin (#610153; BD Biosciences, Franklin Lakes, NJ, USA); mouse monoclonal anti-YAP (SC-101199) and mouse monoclonal anti-phosphotyrosine (PY99, SC-7020) (Santa Cruz, Santa Cruz, CA, USA); rabbit polyclonal anti–α-tubulin (ab18251; Abcam, Cambridge, UK); mouse monoclonal anti-nestin (#33475; Cell Signaling, Danvers, MA, USA); rabbit polyclonal anti EB3 (AB6033; Merck-Millipore, Darmstadt, Germany); AlexaFluor 488-conjugated donkey anti-mouse (A21202); AlexaFluor 488-conjugated goat anti-rabbit (A11008); AlexaFluor568-conjugated goat anti-mouse (A11031); AlexaFluor568-conjugated donkey anti-rabbit (A10042) (Thermo Fisher Scientific, Waltham, MA, USA).
4
Immunohistochemical Analysis of Retinal Cell Types
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Commercial primary antibodies included: 6-11B-1, or mouse monoclonal anti-acetylated α-tubulin (Sigma), rabbit polyclonal anti-GNAT2 (MBL International). The rabbit polyclonal anti-green and blue cone opsin antibodies were gifts from Dr. Thomas Vihtelic (University of Notre Dame) [56 (link)]. The mouse monoclonal anti-rhodopsin antibodies K62-171c and B630 were gifts from Dr. Paul Hargrave (University of Florida) [27 (link)]. Nuclei were stained with Hoechst (Thermo Scientific). Fluorescent secondary antibodies included goat anti-mouse IgG Alexa Fluor 488 and goat anti-rabbit IgG Alexa Fluor 594. Immunogold labeling secondary was either goat anti-mouse IgG 10 nm colloidal gold (Electron Microscopy Sciences) or goat anti-rabbit IgG 10 nm colloidal gold (Electron Microscopy Sciences).
5
Immunohistochemistry of Vestibular and Cochlear Tissues
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Vestibular tissue from mice at P1, P3, and 6 months of age were dissected and then immediately fixed 10 minutes in ice-cold methanol. For labeling of cochlear hair cells, the organ of Corti was removed from mice of different ages, cultured overnight [30 (link)], and then fixed 10 minutes in ice-cold methanol.
Following fixation, vestibular or cochlear tissue was washed in phosphate-buffered saline (PBS), blocked in 2% bovine serum albumin (BSA) for 1 hour, and then incubated with primary antibodies diluted in 2% BSA overnight. Primary antibodies were anti-H340 rabbit polyclonal recognizing SVIL [31 (link)], mouse monoclonal anti-actin (1:100, Clone C4, Millipore, Germany), mouse monoclonal anti-acetylated α-tubulin (1:100, 6-11B1, Sigma, USA), mouse monoclonal anti-β-catenin (1:200, BD Transduction Laboratories, USA), and mouse monoclonal anti-ZO-1 (Invitrogen; Cat. #: 339100). Secondary antibodies were Alexa Fluor 488 chicken anti-rabbit IgG (1:200) and Alexa Fluor 546 goat anti-mouse IgG (1:200, Invitrogen, USA). Alexa Fluor 633 phalloidin (1:50, Invitrogen, USA) was used. Tissue mounted in Vectashield (Vector Laboratories, USA) was imaged on a Leica SP2 or SP8 confocal microscope using a 40 × or 63 × objective (Leica Confocal Software, Leica, Germany).
Following fixation, vestibular or cochlear tissue was washed in phosphate-buffered saline (PBS), blocked in 2% bovine serum albumin (BSA) for 1 hour, and then incubated with primary antibodies diluted in 2% BSA overnight. Primary antibodies were anti-H340 rabbit polyclonal recognizing SVIL [31 (link)], mouse monoclonal anti-actin (1:100, Clone C4, Millipore, Germany), mouse monoclonal anti-acetylated α-tubulin (1:100, 6-11B1, Sigma, USA), mouse monoclonal anti-β-catenin (1:200, BD Transduction Laboratories, USA), and mouse monoclonal anti-ZO-1 (Invitrogen; Cat. #: 339100). Secondary antibodies were Alexa Fluor 488 chicken anti-rabbit IgG (1:200) and Alexa Fluor 546 goat anti-mouse IgG (1:200, Invitrogen, USA). Alexa Fluor 633 phalloidin (1:50, Invitrogen, USA) was used. Tissue mounted in Vectashield (Vector Laboratories, USA) was imaged on a Leica SP2 or SP8 confocal microscope using a 40 × or 63 × objective (Leica Confocal Software, Leica, Germany).
6
Immunofluorescence Staining of Neurons
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Third instar larvae or cultured neurons were fixed according to standard protocols. The following antibodies were used: guinea pig anti-ringer (1:1000) (Mino et al. 2016 (link)), mouse anti-futsch antiserum (1:100; Developmental Studies Hybridoma Bank 22C10), mouse monoclonal antiacetylated α-tubulin (1:100; Sigma T7451), and fluorescence-conjugated secondary antibodies (1:1000; Jackson ImmunoResearch). For microtubule staining, a 1× PHEM (1.8% PIPES, 0.5% HEPES, 0.4% EGTA, 0.04% MgCl2) microtubule stabilization buffer was used for dissections before and after 20 min of methanol fixation at −20°C. Larval body walls were permeabilized with 10% triton x-100 in 5% normal goat serum and 1× PBS overnight at 4°C to improve antibody penetrance for microtubule staining. Images were acquired using a confocal microscope and analyzed in ImageJ software. Z-stacks containing the regions of the cell to be quantified were used to make maximum intensity projections that were then outlined and measured for fluorescence intensity of the respective immunofluorescence in the cell body and axons (proximal and distal).
7
Ciliary Protein Localization Analysis
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The following primary antibodies were used: mouse monoclonal anti–polyglutamylated tubulin (1:1000; clone GT335; cat. no. ALX-804-885; Enzo Life Sciences Ltd); mouse monoclonal anti–acetylated-α-tubulin (1:1500; clone 6-11B-1; Sigma-Aldrich Co LLC, St Louis, Mo); rabbit polyclonal anti–γ-tubulin (1:1000; Abcam Ltd, Cambridge, United Kingdom); affinity purified rabbit polyclonal anti–adenylate cyclase III (ADCY3; 1:500; cat. no. RPCA-ACIII; EnCor Biotechnology Inc, Gainsville, Fla); affinity purified rabbit polyclonal anti-INPP5E (1:200; cat. no. 17797-1-AP; Proteintech Inc, Rosemont, Ill); affinity purified rabbit polyclonal anti-ARL13B (ARL2L1) (1:1000; cat. no. 17711-1-AP; Proteintech Inc); affinity purified rabbit polyclonal anti-IFT88 (1:500; cat. no. 13967-1-AP; Proteintech Inc); rabbit polyclonal anti-Smoothened (1:200; cat. no. bs-2801R; Bioss Inc, Woburn, Mass); mouse monoclonal anti–Ki-67 clone MIB-1 (1:100; cat. no. M7240; Dako UK Ltd, Ely, Cambridgeshire, United Kingdom); mouse monoclonal p53 (1:100; cat. no. NCL-L-p53-Do7; Leica Biosystems Inc, Buffalo Grove, Ill); and rabbit polyclonal anti–dynein axonemal heavy chain 5 (DNAH5; 1:800; cat. no. HPA037470; Sigma-Aldrich Co). Secondary antibodies were Alexa-Fluor568 or 488-conjugated goat anti–mouse IgG and goat anti–rabbit IgG (Thermo Fisher Scientific Inc).
8
Immunohistochemical Profiling of Cartilage
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Primary antibodies were rat monoclonal antibody directed against GAL3 kindly provided by Dr. HakonLeffler (Lund University, Sweden), anti-VDIPEN IgG by Irwin Singer and Ellen Bayne (Merck Research Laboratories, Rahway, NJ, USA), mouse monoclonal anti-acetylated α-tubulin and rabbit polyclonal anti-γ-tubulin from Sigma-Aldrich (Saint-Louis, MO, USA), mouse monoclonal anti-cytochrome C (Bd Pharmingen, Le pont de Claix, France), mouse monoclonal anti-type X collagen (Diagomics, Blagnac, France), rabbit polyclonal anti-ADAMTS-5 (Abcam, Cambridge, UK), mouse monoclonal anti-type 2 collagen (Abcam), and rabbit polyclonal anti-aggrecan (Millipore, Guyancourt, France). Secondary antibodies were goat anti-mouse Alexa-488 and goat anti-rabbit Alexa-568 (Life Technologies, Paisley, UK).
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