5 bromo 2 deoxyuridine brdu

17β-Estradiol, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT), 5-Bromo-2’-deoxyuridine (BrdU), and dimethyl sulfoxide (DMSO) were obtained from Abcam (MA, USA) and Promega (WI, USA).

Free-floating sections (40 μm) were washed in 0.1 M PBS, incubated in 0.1 M PBS containing 5% normal donkey serum and 0.3% TritonX-100 for 1 h, and subsequently incubated overnight with primary antibodies (Nox1, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:500; GFAP, Millipore, Billerica, MA, USA, 1:500; CD11b, Millipore, Billerica, MA, USA, 1:200; Ki67, Thermo Scientific, Fremont, CA, USA, 1:200; 5-bromo-2′-deoxyuridine (BrdU, Abcam, Cambridge, UK), 1:500; doublecortin (DCX), Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:500; NeuN, Millipore, Billerica, MA, USA, 1:1000; active caspase-3, Cell Signaling Technology, Inc. Danvers, MA, USA, 1:500) in 2% normal donkey serum (Vector Lab, Burlingame,CA, USA) in PBS at 4°C and incubated in a 1:200 dilution of Alexa Fluor conjugated donkey anti-rabbit (546) or donkey anti-mouse (647) antibodies (Invitrogen, Grand Island, NY, USA) for 1h at room temperature, washed with PBS, and then mounted sequentially in glass slides using Vectashield (Vector Lab, Burlingame, CA, USA). Mounted slices were evaluated for fluorescence under settings for 546 and 647 emissions on a confocal microscope (Olympus, USA).

Antibodies against GAPDH, GluA1, GluA2, NeuN, and synaptophysin were from Millipore (Billerica, MA, United States). Antibodies against Bip, Robo2, Sema6A, Synapsin-1, Munc18-1, and 5-bromo-2′-deoxyuridine (BrdU) were from Abcam (Cambridge, United Kingdom). Antibodies against γ-protocadherin (γ-pcdh), Rab3A, Rim1, and Munc13-1 were from Synaptic Systems (Gottingen, Germany). Antibody against Slit2 was from Proteintech (Rosemont, IL, United States). Antibody against TrkB was from Cell Signaling (Danvers, MA, United States). Anti-vesicular glutamate transporter 1 (vGluT1) antibody was a gift from Dr. Masahiko Watanabe (Hokkaido University, Sapporo, Japan). Antibodies against both Mea6 and calbindin were from Sigma-Aldrich (St. Louis, MO, United States). Antibodies to β-tubulin and brain-derived neurotrophic factor (BDNF) were from Santa Cruz Biotechnology (Dallas, TX, United States). Goat anti-mouse and anti-rabbit IgG horseradish peroxidase-conjugated were from Thermo Fisher (Waltham, MA, United States). DAPI and Alexa Fluor-conjugated secondary antibody was from Invitrogen (Carlsbad, CA, United States). Protease inhibitor cocktail was from Roche (Mannheim, Germany). Nissl was from Beyotime (Shanghai, China). Other chemicals were from Sigma unless stated otherwise.

The rats were followed up for 30 days (±2 days) after transplantation for their general condition and correction of hyperglycemia. Rats were considered cured if 2 consecutive glucose measures were below 200 mg/dl. In cured animals and those with partial correction of hyperglycemia, an intravenous glucose tolerance test (IVGTT) was performed on day 28, and the scaffold was removed to confirm recurrence of hyperglycemia. The scaffold was fixed in 4% paraformaldehyde overnight and used for immunohistochemical analyses. 5-bromo-2′-deoxyuridine (BrdU; Abcam, Cambridge, United Kingdom) was injected intraperitoneally (i.p.) on days -4, -3, and -2 relative to the scaffold removal day (dose = 500 mg/kg). In one rat per group, micro-computed tomography (micro-CT) was performed at the study end-point to evaluate the AV bundle, as described later.