Anti βiii tubulin

Cell lysates were prepared with Laemmli buffer supplemented with protease inhibitors (complete tablet; Roche) and phosphatase inhibitors (PhosSTOP tablet; Roche) and analysed by Western blotting with the following primary antibodies: anti-γH2AX (Abcam); anti-PTB (Abcam); anti-nPTB (Abcam); anti-βIII Tubulin (Abcam); anti-Tyrosine Hydroxylase (Abcam); anti-GAPDH (Thermo Scientific); anti-MAP2 (Novusbio); anti-DROSHA (Cell Signaling); anti-DICER (Sigma); anti-H2AX (Millipore). Primary antibodies were revealed with peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies (Jackson Immunoresearch Laboratories) and enhanced chemiluminescence system (Super Signal West Pico Pierce or Super Signal West Dura Extended). βIII Tubulin antibody was revealed with alkaline phosphatase-conjugated goat anti-chicken antibody (Santa Cruz) with a colorimetric assay.

We examined the protein expression in synaptodendrosomes and other fractions from homogenate samples by Western blotting. Each band of homogenates was punched and lysed on ice in RIPA buffer (50 mM Tris [pH 7.6], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) with a protease inhibitor cocktail (Cat. No. 11873580001; Roche, Basel, Switzerland). We centrifuged the lysates at 12,000 rpm at 4 °C for 10 min and quantified the proteins in the supernatants using a Pierce BCA Protein Assay Kit (Cat. No. 23225; Thermo Scientific, Waltham, MA, USA). We subjected equal amounts of proteins to SDS-PAGE and probed the membranes with specific antibodies including primary anti-tubulin (Cat. No. Ab195352; Abcam, Cambridge, UK), anti-GFAP (Cat. No. Ab174124; Abcam), anti-synaptophysin (Cat. No. Ab103328; Abcam), anti-PSD95 (Cat. No. Ab122340; Abcam), anti-NeuN (Cat. No. Ab104224; Abcam), and anti-βIII-tubulin (Cat. No. T8578; Sigma-Aldrich) at 1:1000 dilution overnight at 4 °C. We applied secondary antibodies conjugated with horseradish peroxidase to detect reactive bands; these antibodies were developed with the Amersham ECL Plus Detection Kit (GE Health, Chicago, IL, USA).

Alvetex^® Scaffold 3D samples were deparaffinised and rehydrated as previously described. Antigen retrieval was achieved by incubating samples in a 95 °C water bath for 20 min with citrate buffer anhydrous citric acid (Sigma-Aldrich, dissolved in (DI) water). Samples were then permeabilised with 0.1% Triton X-100 in PBS for 20 min and blocked with 1% goat serum (Sigma-Aldrich) and 0.01% Tween in PBS for 30 min. Primary antibody (anti-β—III—tubulin, anti-chondroitin sulphate (Abcam), or anti-brevican (Santa Cruz Biotechnology, Heidelberg, Germany)) was incubated with the samples overnight at 4 °C, followed by three washes in blocking buffer. Samples were then incubated with the relevant secondary antibody (Goat anti-rabbit Alexa Fluor^® 488 or Goat anti-mouse Alexa Fluor^® 488), along with the nuclear dye Hoechst 33,342 for a further 60 min before being washed 3 times for 15 min in blocking buffer and mounted using Vectashield fluorescence mounting media. Confocal images were obtained using the Zeiss 880 and Zeiss 510 confocal microscopes with Zen software, or the Leica SP5 confocal microscope with Leica Application Suite software.

For immunocytochemistry (ICC), cells after 24 h in culture with various treatments were processed for the immunostaining as previously reported^{15 (link)}. Briefly, the PFA fixed cells were permeabilised and blocked followed by overnight incubation with anti β-III tubulin (Abcam, 1:1000) and further with goat anti-mouse IgG conjugated to alexa Flour 488 (Molecular Probes, 1:500). Images were captured using a Motic AE31 microscope.

SFN-Cys and SFN-NAC were purchased from Santa Cruz Biotechnology (USA). PTX (Taxol, as a brand name) was obtained from Selleckchem (USA). Anti-Caspase-3, anti-β-actin, anti-α-tubulin, anti-Tau and protein A/G PLUS agarose were purchased from Santa Cruz Biotechnology (USA). Anti-βIII-tubulin and anti-Caspase-7 were purchased from Abcam (USA). Anti-Hsp70, anti-XIAP, anti-ERK1/2 and anti-pERK1/2 (Thr202/Tyr204) were obtained from Cell Signaling Technology (USA). Anti-Stathmin1 was obtained from Sangon Biotech Co.Ltd. (Shanghai, China). Anti-cleaved-PARP and anti-β-tubulin were purchased from Wanleibio (Shenyang, China). Annexin V-FITC/PI apoptosis assay kit was purchased from NeoBioscience (Shenzhen, China). Recombinant human Caspase-3 was purchased from Sino Biological Inc. (Beijing, China).