Anti beta 3 tubulin antibody

To analyze HTR2B expression after ischemic stroke, formalin-fixed, paraffin-embedded archived brain tissue blocks containing both cortical lesional and peri-lesional areas were selected from 12 stroke patients with a survival post-stroke time ranging from 12 h to 7 days. Death was attributed to complications such as massive edema and brain herniation with rostrocaudal deterioration, or cardiovascular arrhythmias. Written informed consent to autopsy was obtained for each patient from the relatives or their caregivers. Demographic data of patients and controls are given in Table 1.
7μm sections were cut and mounted on positively charged slides (Superfrost®), then dewaxed and rehydrated. Antigen retrieval was performed in heated 1mM citric acid (pH6) and non-specific antigen binding was blocked in 10% goat serum in PBS. Sections were exposed to anti-5-HT2BR antibody (1:50, BD Bioscience) and either anti-AIF1 antibody (1:100, Abcam) or anti-beta III Tubulin antibody (1:1000, Abcam) diluted in 1% goat serum in PBS, then detected using Alexa Fluor 488 and 594 secondary antibodies (Life Technologies). Sections were mounted in ProLong® Gold Antifade reagent with DAPI (Life Technologies) and visualized on a Leica DM6000 fluorescent microscope.

To conduct cytotoxicity assay, SH-SY5Y cells were cultured in medium prepared with DMEM, Ham’s F-12, 2 mM l-glutamine and 1% FBS. Cells were plated in cell carrier-96 clear bottom Perkin Elmer (USA) plates with approximately 2 x 10⁴ cells / well in 100 μl. After 48 hr of cell seeding 0, 12, 24, 36 and 48 hours preincubated Aβ 42 peptide samples such as Aβ 42 alone or with SERPINA3 proteins were mixed with new culture medium containing final 10 ug/mL levofloxacin hemihydrates (Wako, Japan) and added into properly labeled plates. The molar ratio of Aβ 42: SERPINA3 was maintained as final 10 μM: 0.5 μM (20: 1) in assay condition. After another 48 hr of exposure, cytotoxicity effects were determined through LDH assay (LDH-Glo kit, Promega, USA), membrane permeability assay with Propidium Iodide (PI) (Sigma, USA), morphological changes under bright field microscope, Hoechst 33342 (Calbiochem, USA), or DAPI (Sigma, USA) staining to monitor intactness of nucleus. Axonal integrity of microtubules (major component of cell cytoskeleton) was confirmed using anti beta III tubulin antibody (Abcam, USA).

The detection of neuron-specific beta III tubulin-positive cells by flow cytometry was done by first seeding 1 × 10⁴/cm² of mouse iPSCs on 10 cm polystyrene dishes and cultured overnight, then hypoxic stimulus was given by incubation under 5% CO₂, 1% O_2, 94% N₂, and 95% relative humidity at 37 °C for 4 h, and treatment for 4 h with PS80 PBCA NP, HMWC-PS80 PBCA NP, BDNF pDNA, PS80 PBCA NP/BDNF pDNA, HMWC-PS80 PBCA NP/BDNF pDNA were performed followed by a further incubation period under normoxia for 7 days. The cells were fixed with 4% paraformaldehyde and treated with 0.1% Triton-X-100 in DPBS for 10 min. Staining was carried out first with anti-beta III tubulin antibody (1:100, Abcam, Cambridge, UK) under room temperature for 30 min, and with secondary antibodies conjugated to Alexa Fluor^® 488 Alexa (1:1000, Abcam). The fluorescence-labeled cells were placed in 200 µL of DPBS with 1% (w/v) formaldehyde at 4 °C and passed through a polystyrene round-bottom tube with a cell-strainer cap (Corning Inc.). Flow cytometry analysis was carried out by a BD FACSCanto™ II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with Cell Quest software (BD Biosciences).