Anti gpx

QUIN, CUR, o-ophthaldehyde (OPA), NADPH, β-nicotinamide adenine dinucleotide phosphate (NADP⁺), GR, GSH, oxidized glutathione (GSSG), 2,3-naphthalenedicarboxyaldehyde (NDA), H₂O₂, 1-choloro-2,4-dinitrobenzene (CDNB), glucose 6-phosphate, dithiothreitol, bovine serum albumin (BSA), ethylenediamine tetraacetic acid (EDTA), paraformaldehyde (PAF), phenylmethylsulfonyl fluoride (PMSF), protease and phosphatase inhibitors, and primary antibody anti-α-tubulin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Phosphoric acid (H₃PO₄) was obtained from Golden Bell Reagent (Guadalajara, Jalisco, Mexico). Fluoro-Jade B (FJ-B) and polyvinylidene fluoride (PVDF) membrane were obtained from Millipore (Bedford, MA, USA). Primary antibodies anti-Nrf2 (C-20), anti-GR, anti-γ-GCLc, anti-CAT, anti-phospho-ERK1/2, and anti-ERK1/2 were obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Primary antibodies anti-BDNF and anti-GPx were obtained from Abcam (Cambridge, MA, USA). Primary antibodies anti-SOD1 and anti-SOD2 were obtained from Enzo Life Science (Farmingdale, NY, USA). Donkey anti-rabbit, anti-mouse, and anti-goat horseradish peroxidase-conjugate antibodies (secondary antibodies) were from Jackson Immunoresearch Laboratories Inc. (West Grove, PA, USA). Deionized water from a Milli-Q system (Millipore) was used for preparation of solutions.

NP and LP diets were purchased from Pragsoluções Biociências (Jaú, SP, Brazil) and taurine (2-aminoethanesulfonic acid) was from Botica Ouro da Mata Compounding Pharmacy (Campinas, SP, Brazil). MnTMPyP [Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin] pentachloride was from Calbiochem (San Diego, CA, USA). Components of Krebs-Henseleit solution, urethane, serotonin, acetylcholine, sodium nitroprusside, SOD, and apocynin were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Primary antibodies: anti-eNOS from BD Transduction Laboratories (Franklin Lakes, NJ, USA); anti-phospho-eNOS (Ser 1177) and anti-CuZn-SOD from Cell Signaling Technology (Beverly, MA, USA); anti-EC-SOD and anti-Mn-SOD from Enzo Life Sciences (Farmingdale, NY, EUA); anti-p47^phox and anti-catalase from Sigma-Aldrich (St Louis, MO, USA); anti-gp91^phox from Millipore (Millipore Corporation, Bedford, MA, USA); anti-GPX and anti-α-actin from Abcam (Cambridge, MA, USA).

Tissue blocks were processed by the DRI Histopathology Laboratory. Human pancreata and islet graft were fixed in Bouin’s fixative solution (Sigma-Aldrich) for 6h. Human islets were fixed in Bouin’s fixative solution for 1h, dehydrated in 70% ethanol, and embedded in paraffin.
Sections (5μm-thick) were cut on a microtome, air dried overnight, deparaffinized, and rehydrated. After a wash (Optimax Wash Buffer, OWB; Bio-Genex, San Ramon, CA), sections and prepared slides were incubated with Universal Blocker Reagent (UBR; Biogenex),10% human serum for 10min, and washed with OWB. Thereafter, sections were incubated with rabbit anti-catalase (1:100, Rockland, Gilbertsville, PA), rabbit anti-SOD2 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA), or anti-GPX (1:100, Abcam Inc, Cambridge, MA) antibody for 1hr. After washing, tyramide signal amplification (Dako, Carpinteri, CA) in combination with Alexa Fluor 488 antibody (1:200; Molecular Probes, Carlsbad, CA) was used. After staining, sections were incubated with mouse anti-C-peptide (1:100, Abcam Inc, Cambridge, MA), mouse anti-glucagon (1:500, Abcam Inc) for 2 hours at room temperature following incubation with goat anti-mouse Alexa Fluor 647 antibody (1:200, Molecular Probes) with DAPI for 1hr.