Myeloperoxidase mpo

In order assess kidney regeneration, paraffin-embedded sections were submitted to immunohistochemistry after heat mediated antigen retrieval and stained for Proliferating cell nuclear antigen (PCNA, clone PC10) (DAKO, USA) or Myeloperoxidase (MPO) (DAKO, USA), using a Horseradish Peroxidase (HRP)-conjugated secondary antibody EnVision^™+ Dual Link System-HRP (Dako, EUA) revealed with 3,3’-diaminobenzidine (DAB)+ substrate-chromogen (Dako, EUA). In order to evaluate tissue fibrogenesis: Paraffin-embedded sections were submitted to picrosirius staining and subsequent polarization microscopy in order to distinguish collagen fibers. Kidney section images were captured at room temperature (21–25°C) at approximately 10 fields per kidney using an Olympus BX60 microscope and NIS-Elements F capture system (Nikon, Center Valley, PA, USA) or Leica DM 1000 microscope and Leica DFC310 FX (Leica, Wetzlar, Germany) capture system. Positive tissue staining was quantified through the use of Leica Application Suite (Leica, Wetzlar, Germany) or NIS-Elements AR (Nikon, Center Valley, PA, USA) software.

Proteins of interest in renal cortex homogenates were detected by immunoblotting as described previously [36 (link)]. Primary antibodies were against: AS-β, neutrophil gelatinase-associated lipocalin (NGAL), Tfam (GenWay Biotech, San Diego, CA), ED-1 (Serotek, Raleigh, NC), myeloperoxidase (MPO; DAKO, Carpinteria, CA), cleaved caspase-3 (Cell Signaling Technology, Danvers, MA), dynamin-related GTPase protein Drp-1, fissin-1 (Fis-1), mitofusin-1 (Mfn-1), ND3, PGC-1α, PTEN-induced putative kinase 1 (PINK-1) (Santa Cruz Biotech., Santa Cruz, CA) and microtubule-associated protein 1A/1B-light chain 3 (LC3, MBL International, Des Plainers, IL) at concentrations of 1:100 to 1000, and actin (ICN, Costa Mesa, CA) at a concentration of 1:3000. Detection was achieved by chemiluminescence (Pierce Biotechnology, Rockford, IL).

To assess DC phenotype, cells were collected from the bags and washed twice FACS buffer. Thereafter, the cells were incubated at 4°C and stained with the appropriate antibody combinations. Antibodies used for flow cytometry for DC phenotype and purity include: HLA-DR, CD45, CCR7 (from Biolegend), CD11b, CD15, CD16, CD33, CD56, 7-AAD, CD14, CD3, CD83, CD 80, CD11c (all from BD), CD14, CD19, and CD117 (from Beckman & Coulter). For intracellular (IC) stainings, cells were washed with FACS buffer after surface staining and treated with Cytofix/Cytoperm (BD), according to the manufacturer’s protocol, followed by 30 min of incubation at 4°C with the following Abs for the non-DC fraction: cytoplasmic Myeloperoxidase (MPO; Dako). Multiparameter analysis was performed on a FACS Canto II or LSR Fortessa II (BD) flow cytometer. Dead cells were excluded by scatter gating. Analysis was performed using DIVA (BD) or FlowJo software (Tree Star, Inc.).