Ab3623

Tissue and cellular proteins were extracted using RIPA total protein lysate (Aspen, China) following the manufacturer’s instructions. Proteins from each sample were separated by electrophoresis on 8–15% SDS-PAGE gels and transferred onto PVDF membranes (Aspen). After incubation with WB-specific blocking solution (5% skimmed milk powder (AS1033, Aspen, China) diluted in TBST), the PVDF membranes were separately incubated with anti-GLUT1 (1:1000, ab652, Abcam), anti-GLUT4 (1:2000, ab654, Abcam), anti-(extracellular) GLUT3 (1:1000, AGT-023, Alomone), anti-Bax (1:800, sc-7480, Santa Cruz), anti-Bcl-2 (1:800, sc-7382, Santa Cruz), anti-caspase-3 (cleaved) (1:100, AB3623, Millipore), anti-poly (ADP-ribose) polymerase (PARP) (cleaved) (1:1000, 5625, Cell Signaling Technology), and anti-β-actin (1:10,000, Aspen) antibodies at 4 °C overnight. After the blots were washed, they were incubated with HRP-conjugated secondary antibody for 1.5 h and detected by a chemiluminescent imaging system (Tanon, China).

Sections were labelled with IBA-1, 1/2000, ab5076 (Abcam, Cambridge, UK), SY38, 1/2000 (Millipore, Cork, Ireland), AT8, 1/200, MN1020 (Thermo Scientific, MA, USA), Caspase 3, 1/50, ab3623 (Millipore, Cork, Ireland); SY38 sections were treated with 0.2 M boric acid, pH 9 at 65 °C for 30 min then cooled to room temperature prior to quenching. All sections were quenched with 1% H₂O₂/methanol (20 min). Sections were then pre-treated with citrate buffer (pH 6) for 2 × 5 min in the microwave (except SY38). IBA-1 sections were then pre-treated with 0.04% pepsin in 0.1 M HCl for 20 min prior to blocking. Slides were blocked with the appropriate serum. Primary antibodies were incubated overnight at 4 °C. Thereafter the ABC method was used as previously described (Cunningham et al., 2005 (link)) with peroxidase as enzyme, 3,3′-Diaminobenzidine as chromogen and H₂O₂ as substrate. Slides were counterstained using Haemotoxylin (VWR International Ltd, Dublin, Ireland). The DeadEnd Fluorometric TUNEL system (G3250, Promega) was used in conjunction with a biotinylated anti-Fluorescein secondary to visualise apoptotic cells in accordance with manufacturer’s instructions.

Mouse anti-TUJ1 1:1000 (MMS-435P-250, Covance), rabbit anti-CHAT 1:1000 (AB 143, Millipore), mouse anti-NFM 1:500 (MAB1621, Millipore), rabbit anti-ISLET1 1:1000 (20670, Abcam), rabbit anti-GFAP 1:1000 (AB5804, Millipore), rabbit anti-AQP4 1:100 (AB3594, Millipore), rabbit anti-EAAT2 1:100 (ab41621, Abcam), mouse anti-GS 1:100 (MAB302, Millipore), mouse anti-O4 1:1000 (MAB345, Millipore), rabbit anti-IBA1 1:500 (019-19741, Wakochemical), rat anti-CD11b 1:200 (550282, BD pharmingen), mouse anti-MAP2 1:500 (MAB3418, Millipore), mouse anti-TDP-43 1:300 (gift from Dr J-P Julien’s laboratory), mouse anti-NeuN 1:500 (MAB377, Millipore), mouse anti-CNPase 1:200 (NE1020, Millipore), mouse anti-Nestin 1:200 (AB6142, Abcam), mouse anti-SOX2 1:500 (MAB4343, Millipore), mouse anti-Vimentin 1:1000 (AB28028, Abcam), rabbit anti-OLIG2 1:500 (AB9610, Millipore), rat anti-CD31 1:500 (550274, BD Biosciences), rabbit anti-cleaved caspase 3 1:200 AB3623, Millipore), Alexa fluor 488 goat anti-rabbit (A-11008, Life Technologies), Alexa fluor 594 goat anti-mouse (A-11005, Life Technologies)

Anti-NFκB, p65 subunit (MAB3026), anti-tyrosine hydroxylase (TH) and cleaved anti-caspase-3 (AB3623) were purchased from Millipore, Hertfordshire, UK; anti-caspase-2 (ab7979) and anti-caspase-8 (ab52183) were purchased from Abcam, Cambridge, UK.

The following primary antibodies were used for immunoblot analysis or immunohistochemical staining: anti-KIM-1 (ab56015; Abcam, Cambridge, UK), anti-8-OHdG (MOG-100 P; JaICA, Shizuoka, Japan), anti-4-HHE (MHH-030n; JaICA), anti-MnSOD (ab16953; Abcam), anti-PI3K (610045; BD Transduction Laboratories, San Jose, CA, USA), anti-p-AKT (Ser473) (9271 S; Cell Signaling Technology, Danvers, MA, USA), anti-t-AKT (9272 S; Cell Signaling Technology), anti-p-FoxO3a (9466 S; Cell Signaling Technology), anti-t-FoxO3a (2497 S; Cell Signaling Technology), anti-β-actin (A5441; Sigma-Aldrich), anti-COX-IV (A301-899 A; Bethyl Laboratories, Montgomery, TX, USA), anti-Bcl-2 (sc-492; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Bax (DB005; Delta Biolabs, Gilroy, CA, USA), anti-active caspase-9 (9505 S; Cell Signaling Technology), and anti-active caspase-3 (AB3623; Millipore Corporation, St. Charles, MO, USA).

Primary antibodies used for confocal microscopy or immunoblot analysis were as follows: anti-insulin (I2018, Sigma-Aldrich; 18-0067, Invitrogen, Camarillo, CA, USA), anti-glucagon (8233; Cell Signaling Technology, Danvers, MA, USA), anti-active caspase-3 (AB3623; Millipore Corp.), anti-8-OHdG (MOG-020P; JaICA, Shizuoka, Japan), anti-p62 (AB56416, Abcam, Cambridge, UK; GP62-C, Progen Biotechnik GmbH, Heidelberg, Germany), anti-ubiquitin (PA5-17067; Thermo Fisher Scientific, Rockford, IL, USA), anti-LC3B (L7543, Sigma-Aldrich; AB168831, Abcam; ALX-803-080, ENZO Life Sciences, Inc., Farmingdale, NY, USA), anti-LAMP-2A (3900-100; BioVision Inc., Milpitas, CA, USA), and anti-DDK (TA50011-100, OriGene Technologies).

For 3D culture cells were fixed with 4% (v/v) paraformaldehyde (PFA) in PBS for 20 minutes and then washed once with PBS. 2D cultures were maintained until the cells attained 70% confluency and then were fixed with 4% (v/v) PFA in PBS for 20 minutes and then washed once with PBS. To prevent the nonspecific adsorption of antibodies, the cells were incubated with 0.1% BSA and 10% goat serum for 1 hour. The primary antibodies (anti-Giantin, Abcam ab24586, 1/500; anti-caspase 3 active, Millipore AB3623, 1/300; anti-beta4-integrin, Millipore MAB1964, 1/250; anti-centrosome, kind gift from M. Thery, CEA Grenoble, F) were dissolved in PBS⁺, Tween20 0.05%, and 5% goat serum. The cells were incubated for 1 hour with the appropriate antibody and then washed 4x with for a total of 45 minutes. The cells were then incubated with secondary antibody (Jackson, dilution 1/500 in PBS⁺, Tween20 0.05%, and 5% goat serum). Cells were subsequently washed 4 times for 15 minutes with PBS. Nuclei and actin were stained as described hereafter.