βiii tubulin

Primary DRG neurons were fixed with 4% paraformaldehyde in cytoskeletal preservation buffer (10 mM MES, 138 mM KCl, 3 mM MgCl₂, 2 mM EGTA, 320 mM sucrose, pH 6.1) for 15 minutes at room temperature. Samples were washed with PBS (3 times for 5 minutes each) and permeabilized in PBS with 0.3% Triton X-100 for 5 minutes. Cells were rinsed briefly in PBS and blocked for 1 h at room temperature with 5% BSA in PBS-T (0.1% Tween-20 in 1X PBS). Samples were incubated overnight in 1% BSA in PBS-T at 4°C with the following primary antibodies: β-III-tubulin (1:1,000, BioLegend), Pum1 (1:200, Bethyl), Pum2 (1:200, Bethyl), GSK-3β (1:100, Cell Signaling). Samples were washed with PBS-T (3 times, 5 minutes each) and incubated for 1 hour at room temperature with fluorophore-conjugated Alexa secondary antibodies (1:1,000, Thermo Fisher Scientific) and/or fluorescence conjugated β-III-tubulin (1:500, BioLegend). Samples were washed with PBS (3 times, 5 minutes each), and mounted with ProLong Diamond antifade reagent (Thermo Fisher Scientific). Imaging was carried out using an EC Plan-Neofluar 40×/1.3 objective on an Axio-Observer.Z1 microscope equipped with an AxioCam MRm Rev. 3 camera or on an LSM 800 confocal microscope (Zeiss, Thornwood, NY).

Tissue was sectioned at 10 μm and following deparaffinization (3 day only) samples underwent antigen retrieval in Tris EDTA buffer at pH 9 for 15 min at 95°C. A standard IHC protocol was followed [17 ], in brief, samples were permeabilized in 0.1% Triton X-100 for 7 minutes, rinsed, then incubated in 100% FBS for 1 hour, rinsed, then incubated in primary antibody overnight at 4°C, rinsed three times, then incubated in secondary antibody for one hour at room temperature, rinsed three times, then incubated in 10 μM DAPI for 5 min, rinsed, and mounted with Prolong Gold (Thermo-Fisher Scientific). Antibodies used are as followed: NSC marker Nestin 1:20 (DSHB RAT-401). Neuronal marker βIII tubulin 1:400 (BioLegend, 801201), neutrophil marker myeloperoxidase (Thermofisher) 1:200, macrophage marker ED-1 clone (Sigma Aldrich) 1:150 with 1:500 secondary Alexa Fluor 488 donkey anti-rabbit and Alexa Fluor 647 goat anti-mouse (ThermoFisher Scientific). Slides exposed to the whole process except for primary antibodies were used as negative controls throughout. Imaging was performed using a Nikon A1 confocal system or Olympus FV1000 with gain and laser power consistent for each fluorescent secondary antibody used. ImageJ was used to threshold images and quantify positive staining normalized to DAPI.