Anti acetylated tubulin

Embryos or sectioned embryonic slices were incubated in blocking solution (5% BSA + 2% DMSO in TBS + 0.1% Triton X-100) at room temperature for 30 min to block nonspecific binding. TSA-staining was performed with the following primary antibodies: anti-acetylated tubulin (Sigma-Aldrich), anti-phospho-histone H3 (Abcam), anti-fibronectin (DSHB), and anti-MHC (DSHB) at a 1:300 or 1:1,000 dilution for 3 hr at room temperature. The samples were rinsed with TBST (TBS + 0.1% Triton X-100). Labeling was performed with HRP-conjugated secondary antibodies (Sigma-Aldrich) or HRP-GST-ABD at 1:1,000 dilution for 2 hr. For the HRP-GST-ABD, the indicated primary antibody was pre-incubated with HRP-GST-ABD in a 1:1 ratio (0.8 ng/μl each) and the tissue samples were incubated with the mixture. Tyramide labeling was performed using tyramide Alexa-488 in amplification buffer with 0.00015% hydrogen peroxide. The mixtures were incubated at room temperature for 2 min and the tissue samples were rinsed with TBST. The nuclei were stained with DAPI (Abcam). All sectioned slices were run through washes in 100% methanol before clearing in BA:BB (benzyl alcohol/benzyl benzoate, 1:2) and mounting on slides for imaging. Images were captured using a confocal microscope (LSM700).

Cryosections were prepared from adult enucleated eyes or whole larvae and immunostained as previously described (10 (link),52 (link)). Primary antibodies used were 4D2 (1:200; Abcam, Cambridge, UK; ab98887), anti-UV opsin, anti-blue opsin, anti-red opsin (1:200; gifted by Professor David Hyde, University of Notre Dame), anti-usherin-C (1:500; Novus Biological, Littleton, CO, USA; 27 640 002), anti-usherin-N (1:200; gifted by Dr Jennifer Phillips, University of Oregon), anti-LC3 (1:200; Thermo Fisher Scientific, Waltham, MA, USA; PA1-16930), anti-centrin (1:500; Merck-Millipore; 04-1624) or anti-acetylated tubulin (1:200; Sigma-Aldrich, St Louis, MO, USA; T7451). Primary antibodies and appropriate secondary Alexa Fluor antibodies (1:500; Thermo Fisher Scientific) were diluted in antibody solution. The slides were imaged using a Leica LSM 700 confocal microscope or a Zeiss Axio Imager fluorescence microscope equipped with an Axiocam MRC5 camera. Rhodopsin levels were quantified using ImageJ. For this, all pictures were taken using the same settings after which the region of interest (adult retina: rod outer segments, larval retina: inner segment and ONL) was defined manually and assessed using the mean pixel intensity measurement.

Embryos were processed for WISH, cryo-sectioning, and immunohistochemistry as before (Korzh et al., 1998 (link)). The following antibodies were used: mouse monoclonal anti-GFP antibodies (clone B-2, Santa Cruz Biotechnology, 1 mg/ml), anti-acetylated tubulin (Sigma–Aldrich, T6793, 2 mg/ml), AFRUMA antibodies (1:500; kindly provided by Prof. J. M. Grondona; (Rodríguez et al., 1984 (link); López-Avalos et al., 1997 (link)) and goat anti-mouse/Alexa Fluo488 (Molecular Probes, 2 mg/ml).

Transduced HeLa, NHEK, and serum-starved hTERT-RPE1 cells were washed with PBS and harvested in the FLAG lysis buffer. Supernatants were incubated with 30 μL anti-FLAG-beads (ANTI-FLAG^TM M2 Affinity Gel) overnight at 4 °C and 1 h at RT on a rotator to pool-down ARP-T1-FLAG proteins. Beads were separated by centrifugation for 3 min at 5000 × g, washed five times with TBS, and eluted in 23 μL TBS and 7 μL 5x SDS-sample buffer at 85 °C for 5 min. ARP-T1 precipitation was confirmed using ARP-T1 antiserum (1:2000, GP-SH6), and co-precipitated proteins were analyzed using anti-acetylated tubulin (1:1000, T6793, Sigma-Aldrich), anti-TCP8 / TCP1 theta (1:500, PA5-30403, Thermo Scientific), anti-HSC70 (1:200, PA5-27337, Thermo Scientific), anti-BAG2 (1:100, PA5-30922, Thermo Scientific), anti-gamma tubulin (1:500, ab11316, Abcam), anti-EDH4 (1:1000, Dr. Plomann’s lab), anti-septin 2 (1:2000, HPA018481, Sigma-Aldrich), anti-septin 9 (1:2000, HPA042564, Sigma-Aldrich).

2 μm-thick formalin-fixed paraffin-embedded sections were deparaffinised, rehydrated and permeabilised (0.1% Triton X-100 in PBS for 8 min). Antigen retrieval was performed with Tris-buffered saline (TBS, pH 9 at 96 °C for 20 min). Slides were incubated with monoclonal mouse anti-Acetylated-tubulin (1:4000, Sigma Aldrich, T7451; St. Louis, MO, USA) or polyclonal rabbit anti-Pericentrin (1:100, Abcam, ab4448; Cambridge, UK) in a dark humidified chamber. The appropriate secondary antibody: donkey anti-mouse IgG Alexa Fluor 594 (1:1000, ThermoFisher, R37115; Waltham, MA, USA) or donkey anti-rabbit IgG Alexa Fluor 488 (1:1000, ThermoFisher, A-21206; Waltham, MA, USA) were applyied. Sections were washed with PBS, counterstained with DAPI (1 μg/mL, Sigma-Aldrich) and mounted with fluorescence mounting medium (DAKO, S3023). Samples were visualised with a fluorescence microscope (Olympus BX1 with DP70 Digital Camera System) and analysed with DP Controller Software and FIJI ImageJ software^{58 (link)}.