To depolymerize actin and tubulin, egg chambers were treated with 100 μM Latrunculin A (Abcam) and 50 mg/ml colcemid (Santa Cruz Biotechnology), respectively, in Schneider's medium for 1.5 h at 25°C. Control egg chambers were incubated in Schneider's medium for 1.5 h at 25°C. Egg chambers were fixed in 4% formaldehyde, 2% Tween 20 in PBS for 20 min and then washed 3×5 min in 0.1% Tween in PBS. Egg chambers were then incubated with a 1:500 dilution of Phalloidin-iFluor 405 (Abcam) and anti-α-Tubulin−FITC antibody (Merck) in PBS, 0.5% BSA at 37°C for 1 h with shaking. Samples were washed for 6×10 min in 0.2% Tween in PBS buffer, mounted in Vectashield with DAPI (Vector Laboratories) and examined via confocal microscopy (Leica SP8).
Anti α tubulin fitc antibody
Manufactured by Merck Group
Sourced in United States
Anti-α-tubulin-FITC antibody is a fluorescently labeled antibody that binds to the alpha-tubulin protein component of the cytoskeleton in cells. It is used as a tool in cell biology research for the detection and visualization of microtubules.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Merck Group (3 mentions)
Phalloidin ifluor 405, by Abcam (2 mentions)
Vectashield with dapi, by Vector Laboratories (2 mentions)
Las image analysis software, by Leica (2 mentions)
Dmi4000b, by Leica (2 mentions)
Alexa fluor 488 goat anti rabbit antibody, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 goat anti rabbit antibody, by Thermo Fisher Scientific (2 mentions)
Fitc phalloidin, by Merck Group (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Colcemid, by Santa Cruz Biotechnology (1 mentions)
Latrunculin a, by Abcam (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Bimecy 0500, by Eurogentec (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Axio observer z1 inverted microscope, by Zeiss (1 mentions)
Confocal laser scanning microscopy, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Triton x 100, by Solarbio (1 mentions)
780 meta, by Zeiss (1 mentions)
Ultraview vox confocal imaging system, by PerkinElmer (1 mentions)
27 protocols using anti α tubulin fitc antibody
1
Depolymerization of Actin and Tubulin in Egg Chambers
2
Visualizing Cytoskeleton Depolymerization
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To depolymerise actin and tubulin, egg chambers were treated with 100 μM Latrunculin A and 50 mg/ml Colcemid respectively in Schneider's medium for 1.5 hours at 25 °C. Control egg chambers were incubated in Schneider's medium for 1.5 hours at 25 °C. Egg chambers were fixed in 4% Formaldehyde, 2% Tween 20 in PBS for 20 min and then washed 3 x 5 min in 0.1 %Tween in PBS. Egg chambers were then incubated with a 1:500 dilution of Phalloidin-iFluor 405 (Abcam) and anti-α-Tubulin-FITC antibody (Merck) in PBS, 0.5% BSA at 37°C for 1 hour with shaking. Samples were washed 6 x10 min in 0.2% Tween in PBS buffer, mounted in Vectashield with DAPI (Vector Laboratories) and examined via confocal microscopy (Leica SP8).
3
Visualizing Microtubules in Cultured Cells
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Cells were cultured on chamber slides and treated with LNS8801 for 24 hours. Cells were fixed in 4% formaldehyde, permeabilized in 0.05% tween, blocked with 2% FBS, and incubated with an anti-α-tubulin FITC antibody (Sigma-Aldrich) for 1 hour. Cells were washed with PBS and stained with 4,6-diamino-2-phenylindole (DAPI). Images were acquired on Nikon A1 confocal microscope at 60× magnification and visualized with Fiji ImageJ.
4
Visualizing Oocyte Spindle Morphology
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Oocytes cultured for 42 h were fixed in 4% paraformaldehyde for 30 min, permeabilized in 0.5% Triton X-100 (v/v in PBS) for 30 min, blocked in 5% BSA (w/v in PBS) for 2 h, and incubated with anti-α-Tubulin-FITC antibody (1:800 dilution, F2168, Sigma) for 1 h to visualize the spindle morphology (Nie et al., 2017 (link)). After three washes and DNA staining with DAPI, oocytes were mounted and captured by using a confocal microscope and an oil objective with the same scanning settings. All steps were performed at room temperature. Normal morphologies of chromosomes and spindles were classified as previously described (Nie et al., 2017 (link)): Type I, chromosomes are aligned on the midline with spindle-shaped microtubules alongside; Type II, punctate chromosomes are detected in cobweb-shaped microtubules at the metaphase plate. Disorganized spindles containing condensed chromatin were regarded as abnormal Type III, and multipolar spindles with misaligned chromosomes were regarded as abnormal Type IV.
5
Mitotic Spindle Morphology Assay
6
Oocyte cryoprotectant composition analysis
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Fresh MII oocytes and vitrified-thawed oocytes from groups of 10% DMSO plus 10% EG with 2.00 mol/L L-proline, 10% DMSO plus 7.5% EG with 2.00 mol/L L-proline, 7.5% DMSO plus 10% EG with 2.00 mol/L L-proline, 7.5% DMSO plus 7.5% EG with 2.00 mol/L L-proline, and 15% DMSO plus 15% EG without L-proline were fixed in 4% paraformaldehyde for 30 min. For 5-methylcytosine (5-mC) staining, DNA of the oocytes was denatured with 4.00 mol/L HCl at 25°C for 10 min and subsequently neutralized by treatment with 100 mmol/L Tris-HCl buffer (pH 8.0) for 10 min. After washing, samples were blocked in blocking solution (3% bovine serum albumin [Sigma] prepared in PBS) at 25°C for 1 h, and then incubated with an anti-5-mC antibody (1:50; BIMECY-0500; Eurogentec, Belgium) for 2 h at 25°C. After washing three times, oocytes were incubated with Alex Fluor 568-conjugated goat anti-mouse IgG (1:500, A-11004; Invitrogen, USA) for 2 h at 25°C. After rinsing, samples were incubated with a monoclonal anti-α-tubulin-FITC antibody (1:100, F2168; Sigma, USA) for 1 h. Finally, nuclei were counterstained with 10 mg/ml Hoechst 33342 (Molecular Probes, H3570, Life Technologies, USA) for 15 min. For negative controls, the primary antibody was omitted. All groups of oocytes were observed with a confocal laser scanning microscope (LSM710 Carl Zeiss, Oberkochen, Germany).
7
Antibody Sources for Tmod3 and Tpm3
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Goat polyclonal antibody against human Tmod3 (G-16, sc-19205) was acquired from Santa Cruz Biotech (Santa Cruz, CA, USA). A mouse monoclonal antibody against Tpm3 (CG3)61 (link) was obtained from the Developmental Study Hybridoma Bank at the University of Iowa. An Alexa-Fluor-488-conjugated rabbit anti-goat-IgG antibody was purchased from Invitrogen (Carlsbad, CA, USA), and a mouse monoclonal anti-α-tubulin–FITC antibody was obtained from Sigma (St Louis, MO, USA).
8
Dual Immunofluorescence Assay for AhR-RelB Interaction
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MEF cells expressing WT or Q377A AhR or cDC2 stimulated as indicated in Figure 6 D, were fixed 10 minutes with 4% PFA, permeabilized, blocked in BSA 5% in 1 X PBS. Duolink® (#DUO92008, Sigma-Aldrich) was performed, according to the manufacturer's protocol. In brief, primary antibodies, sheep anti-AhR (#AF6697, R&D System) and mouse anti-RelB (clone D-4, #sc-48366) were conjugated with either PLUS (#DUO92009, Sigma-Aldrich) or MINUS (#DUO92010, Sigma-Aldrich) oligonucleotide, to create PLA probes. Samples were incubated overnight at 4°C and, subsequently, ligase solution was added for 30 minutes. The signal was amplified with amplification polymerase solution at 37°C for 100 minutes. Then, a counterstain was carried out using a directly labelled FITC anti-alpha tubulin antibody (Anti-α-Tubulin-FITC antibody, #DM 1A Sigma-Aldrich). Nuclei were counterstained with 4’, 6’-diamidino-2phenylindole (DAPI) (#DUO82040, Sigma-Aldrich). Images were captured using a Zeiss Axio Observer Z1 inverted microscope, equipped with Apotome filter and Axiocam MRm camera detection system Zeiss (Castelli et al., 2020 (link)).
9
Immunofluorescent Imaging of Oocyte Cytoskeleton
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Denuded matured oocytes underwent fixation using 4% (w/v) paraformaldehyde (PFA) for a duration of 45 min at room temperature (RT). Subsequently, the oocytes were made permeable using 0.5% Triton X-100 (Solarbio, China) at RT for an hour. After being blocked in 3% BSA solution for 1 h at RT, the oocytes were reacted with anti-α-tubulin-FITC antibody, (Sigma, USA, 1:2000) overnight at 4 °C. Then, they were washed with 0.1% Tween 20 three times. Finally, the oocytes were dyed with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA) for 6 min, and the fluorescent images were taken with laser scanning confocal microscopy (Leica, Wetzlar, Germany).
10
Immunofluorescence and Mitochondrial Staining of Oocytes
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Immunofluorescence was performed by using previously published methods [45 (link)]. Briefly, oocytes were fixed for 30 min at room temperature in 2% formaldehyde supplemented with 100mM HEPES, 50mM EGTA, 10mM MgSO4, 0.2% Triton X-100 (pH=7, titrated with KOH). Then they were treated with PBS, 0.1% Triton X-100 overnight at 4oC and incubated with anti-α-tubulin-FITC antibody (F2168, Sigma, USA) (1:1000 in PBS with 0.1% Triton X-100 and 3% BSA) overnight at 4oC. Chromosomes were stained with DAPI for 15 min. The oocytes were mounted on glass slides and examined with a laser scanning confocal microscope (Zeiss 780 META).
For staining active mitochondria, eggs were incubated for 30 min at 37°C in M2 medium containing 200nM MitoTracker ® Red CMXRos (M7512, Invitrogen, USA). After washing 3 times, eggs were stained with Hoechst 33342 (10mg/ml) for 15 min. Finally, eggs were mounted on glass slides and examined with a Perkin Elmer UltraVIEW VOX confocal Imaging System.
For staining active mitochondria, eggs were incubated for 30 min at 37°C in M2 medium containing 200nM MitoTracker ® Red CMXRos (M7512, Invitrogen, USA). After washing 3 times, eggs were stained with Hoechst 33342 (10mg/ml) for 15 min. Finally, eggs were mounted on glass slides and examined with a Perkin Elmer UltraVIEW VOX confocal Imaging System.
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