Mouse monoclonal anti acetylated tubulin antibody

For whole-mount immunofluorescence staining, zebrafish embryos and larvae were fixed in 4% paraformaldehyde in phosphate-buffered saline overnight at 4 °C and then permeabilized in cold acetone at −20 °C. In addition, 4-dpf-old larvae were permeabilized with collagenase A (Roche Diagnostics, 1 mg ml⁻¹) for 90 min. Embryos/larvae were blocked in 5% horse serum in phosphate-buffered saline containing 0.1% Tween-20 (PBT). Embryos/larvae were incubated in blocking solution containing primary antibody overnight at 4 °C followed by washing several times with PBT and incubation with secondary antibody (anti-rabbit Alexa Fluor 594 and anti-mouse Alexa Fluor 488, Invitrogen). The primary antibodies used in this study were a polyclonal rabbit anti-MBP antibody (1:50 dilution, a kind gift from Prof. William Talbot, Stanford University) and a mouse monoclonal anti-acetylated tubulin antibody (1:500 dilution, Sigma) to label axon tracts. Combinational staining of myelin, neurons and nuclei in zebrafish embryos was performed using BrainStain Imaging Kit (Molecular Probes, B34650). Stained embryos/larvae were imaged with a Zeiss Axio Imager microscope. Immunofluorescence images were collected using a Zeiss Axio Imager Z1 fluorescence microscope equipped with Zeiss Apotome 2 (Zeiss, Germany) in AxioVision Rel 4.9 software.

Eggs were subjected to immunofluorescence staining to detect microtubules, as previously described23 (link). The primary antibodies were mouse monoclonal anti–acetylated tubulin antibody (1.0 mg/ml) (Sigma–Aldrich, St. Louis, MO, USA) and mouse monoclonal anti–β-tubulin antibody (2.0 mg/ml) (Sigma–Aldrich), and the secondary antibody was Alexa Fluor® 488–conjugated goat anti–mouse IgG (2.0 mg/ml) (Life Technologies, Carlsbad, CA, USA). Supplier data sheets for all three antibodies recommend that they should be used for immunofluorescence staining at a dilution of 1:200–1:1,000, and previous studies often used them at these concentrations5 (link)24 (link). For these experiments, primary antibodies were diluted 1:1,000, 1:2,000, 1:4,000, 1:8,000, or 1:16,000, and the dilution of the secondary antibody was fixed at 1:1,000. Eggs were mounted in VECTASHIELD® Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA) to allow observation of chromosomes and prevent fading. For each dilution, we stained six eggs.

The quantitative analysis of nerve abundance was performed on regenerating limb and flank sections using the Mouse Monoclonal Anti-Acetylated Tubulin antibody (1:200 dilution – Sigma-Aldrich), followed by the Goat-Anti-Mouse IgG Alexa Fluor 488 (1:200 dilution – Abcam, Cambridge, MA) secondary antibody. They were co-stained with Rhodamine phalloidin and DAPI as a general tissue stain, and to provide positional context for the location of the axon bundles. Limb nerve abundance was quantified by determining the percentage of limb area that is positive for Anti-Acetylated Tubulin staining. To determine limb-bound axon bundle size, the sum of the area of axon bundles from DRGs 3, 4, and 5 were quantified as they emerged from the skeletal muscle surrounding the spine (Figure 5—figure supplement 1).

Antibodies were used at the following concentrations: rabbit anti-Smo antibody [11] (link) 1∶1000, mouse monoclonal anti-acetylated tubulin antibody (Sigma) 1∶2000, mouse anti-beta tubulin (Developmental Studies Hybridoma Bank) 1∶5000, secondary antibodies (Jackson ImmunoResearch Laboratories) 1∶500.