Mouse monoclonal anti alpha tubulin

Samples were fixed at different time points in fresh 4% paraformaldehyde (PFA). The suspension was allowed to adhere onto poly-l-lysine coated slides overnight at 4°C. The slides were washed once with PBS and immunocytochemistry performed per manufacturer's instructions for rabbit monoclonal anti-myc 1:200 (Cell Signaling). Mouse monoclonal anti-alpha tubulin 1:500 (Sigma) was used simultaneously or independently using the same protocol. Secondary antibodies Alexa 488-conjugated anti-rabbit IgG and Alexa 568 conjugated anti-mouse IgG for fluorescence detection were used at 1:1000 (Molecular probes). The slides were mounted in Vectashield with DAPI (Vector Labs). Parasites were visualized on a Leica SP5 confocal microscope and acquired and analysed with the LAS AF Lite software (Leica). For quantification, samples were visualized on a Leica DMR microscope and imaged with the Zeiss AxioCam HRC and Axiovision software respectively. Cell nuclei diameters were measured using ImageJ and statistical analysis performed using Student's t-test.

Taxol/paclitaxel were purchased from Sigma-Aldrich, Inc., and the dry powder was prepared for 100 μM stock solutions in DMSO. Tissue culture flasks (trade mark Falcon), tissue culture media, trypsin, and 100X antibiotic-antimycotic solution (Cellgro, Mediatech, Inc) were purchased from VWR Inc. (Springfield, NJ). Alexa Fluor 488 and 555 conjugated secondary antibodies were purchased from Life Sciences Inc. (Eugene, Oregon) for use in immunofluorescence microscopy. Primary antibodies: anti-Lamin A (1:400, H-102, rabbit polyclonal IgG); mouse monoclonal anti-Lamin B (1:300, sc373918); and goat polyclonal anti-Lamin B (1:400, sc6216) were from Santa Cruz Biotechnology Inc.; rabbit polyclonal anti-Lamin B1 (1:1000, ab16048) and mouse monoclonal anti- αTubulin (1:500, 66,031) were from Abcam and Proteintech, respectively. Mouse monoclonal anti-alpha-tubulin, were purchased from Sigma-Aldrich, Inc. (St. Louis, MO). More details of the reagents used are also reported previously [59 (link)–62 (link)].

The following primary antibodies were used: rabbit polyclonal anti-MURC/cavin-4 for immunohistochemical analysis (Code: HPA020973, Sigma-Aldrich, Milan, Italy); goat polyclonal anti-MURC/cavin-4 for immunoblotting analysis (Code: SC-163021, Santa Cruz Biotechnology, Dallas, TX, USA); rabbit polyclonal anti-MURC/cavin-4 for immunofluorescence analysis (as described in [52 (link)]); mouse monoclonal anti-Cav-3 (Code: 610420, BD Transduction Laboratories, Buccinasco, Milan, Italy); rabbit anti-myogenin (Santa Cruz Biotechnology, Dallas, TX, USA, 1:500 dilution); mouse monoclonal anti-MHC (Code: SC-32732, Santa Cruz Biotechnology, Dallas, TX, USA); mouse monoclonal anti-total and—phosphorylated ERK1/2 (extracellular regulated kinase 1/2) (Tyr204) (Code: SC-135900 and SC-7383, Santa Cruz Biotechnology, Dallas, TX, USA); mouse monoclonal anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (Code: MAB374, Millipore, Darmstadt, Germany) and mouse monoclonal anti-alpha-tubulin (Code: T5168, Sigma-Aldrich, Milan, Italy).

Cells were fixed with either chilled 100% methanol for 10 minutes or 4% paraformaldehyde solution at room temperature for 15 min and subsequently permeablized with 0.2% Triton‐X in PBS for 5 min. Cells were blocked for 1 h using 2% normal goat serum in PBS prior to overnight incubation with primary antibody solution at 4°C: mouse monoclonal anti‐alpha tubulin (Sigma, catalogue no. T6199, 1:400), rabbit polyclonal anti‐paxillin (Abcam, catalogue no. ab191007, 1:200), rabbit polyclonal anti‐GEF‐H1 (Abcam, catalogue no. ab155785, 1:200), rabbit polyclonal anti‐myosin IIA (Sigma, catalogue no. M8064, 1:200), and rabbit polyclonal anti‐Piezo1 (Novus Biologicals, catalogue no. NBP1‐78446, 1:200). Samples were washed with PBS three times and incubated with Alexa Fluor‐488 or −594 secondary antibodies (ThermoFisher) for 1 h at room temperature followed by PBS washing. F‐actin was stained using Alexa Fluor 594 phalloidin conjugated antibody (ThermoFisher). Cell nucleus was stained using Hoechst dye (1:1000, ThermoFisher).
Images were acquired using either wide‐field Olympus live‐EZ microscope equipped with photometrics CoolSNAP K4 camera or W1 live‐SR spinning disk microscope equipped with photometrics Prime 95B sCMOS camera.

HEp-2 cells grown overnight on coverslips were transfected with 1 μg of the desired DNA plasmid using Turbofect (Thermo Scientific) as recommended by the manufacturer. After the desired time (see Figure legends) at 37°C with 5% CO₂, cells were washed with 1x PBS, fixed in 3.7% paraformaldehyde. For staining of microtubule, γ-tubulins or peroxisomes, cells were permeabilized with 0.1% Triton X-100, incubated with a monoclonal mouse anti-alpha tubulin (Acris), a monoclonal mouse anti-γ-tubulins (Sigma-Aldrich) or a monoclonal mouse anti-PMP70 primary antibodies (Sigma-Aldrich), and then with proper labeled secondary antibodies. To stain lipid droplets, fixed HEp-2 cells were permeabilized with 0.5% saponin and stained with Bodipy 493/503 (Molecular Probes). Actin staining of human cells with rhodamine-phalloidin was performed as described by the manufacturer (Molecular Probes). DNA was stained with Dapi. Microscopy was carried out using either spinning-disk confocal microscopes (Carl Zeiss).

In addition to assessing for meiotic maturation, a subset of oocytes that had reinitiated meiosis (GVBD + MII) underwent preliminary evaluation for spindle structure and chromosome alignment.
The oocytes were fixed and stained as described by Lenie et al. (2008) (link). Oocyte spindles were stained by sequentially incubating the oocytes on monoclonal mouse anti-alpha-tubulin (Sigma) and Alexa Fluor 488-polyclonal goat anti-mouse antibody (Molecular Probes). Chromosomes were stained with Nucblue_® (Molecular Probes). Antibody dilution and intermediary washing steps were performed with a wash-block solution (PBS containing 0.02% sodium azide, 0.2% milk powder, 2% normal goat serum (NGS), 1% BSA, 0.1 M glycine, and 0.01% Triton X-100). Incubation was carried out at 37ºC. Stained oocytes were viewed on an inverted microscope (Motic) equipped with a fluorescent lamp and appropriate filters; images were recorded with software Motic Images Plus 3.0.