Azolium chloride (TTC) staining of tissues 48 hours after TBI. The animalsAzolium chloride (TTC) staining

Azolium chloride (TTC) staining of tissues 48 hours after TBI. The animals
Azolium chloride (TTC) staining of tissues 48 hours after TBI. The animals were decapitated, and the brain was sectioned into four 2-mm coronal sections by using a mouse brain matrix. The brain slices were then stained with 2 TTC at 37 for 30 min and photographed on the anterior surface of each section with a scale bar. The areas of injured brain were delineated by examining differences between the ipsilateral and contralateral regions in the center slice of injured brain and measured by using NIH Image software. http://rsb.info.nih.gov/nih-image/about.html.Dohi et al. Journal of Neuroinflammation 2010, 7:41 http://www.jneuroinflammation.com/content/7/1/Page 3 ofTable 1 Antibodies used for immunoblotting (IB) and immunohistochemistry (IHC)Antibody Primary antibody (clone #) gp91phox p22phox iNOS Ym1 GAPDH (6C5) b-Actin (AC-74) CD11b (5C6) GFAP (G-A-5) NeuN 3-NT Secondary antibody (conjugation) Mouse IgG (HRP) Rabbit IgG (HRP) Rabbit IgG (biotinylated) Mouse IgG (Alexa 546) Rabbit IgG (Alexa 488 or 546) Rat IgG (Alexa 546) Mouse IgG Rabbit IgG Rabbit IgG Mouse IgG Rabbit IgG Rat IgG Sheep Donkey Goat Goat Goat Goat GE Healthcare Bioscience (Little Chalfont, UK) GE Healthcare Bioscience (Little Chalfont, UK) Santa Cruz Biotechnology (Santa Cruz, CA) Molecular Probes (Eugene, OR) Molecular Probes (Eugene, OR) Molecular Probes (Eugene, OR) NA931 NA934 SC-2040 A11030 A11034 or 11035 A11081 2,000 3,000 200 400 400 400 Human gp91 Human p22 Mouse iNOS Mouse Ym1 Rabbit GAPDH Mouse b-Actin Mouse CD11b Mouse GFAP Mouse NeuN Nitrated KLH Rabbit Rabbit Rabbit Rabbit Mouse Mouse Rat Mouse Mouse Rabbit See ref 18 See ref 18 Transduction Laboratories (Lexington, KY) StemCell Tech (Vancouver, BC, Canada) Chemicon International (Temecula, CA) Sigma (St Louise, MO) Serotec (Oxford, UK) Sigma (St Louise, MO) Chemicon International (Temecula, CA) Upstate Biotechnology (Lake Placid, NY) N32030 01404 MAB374 A5316 MCA711 G3893 MAB377 06-284 4,000 (IB) 200 (IHC) 3,000 10,000 1,000 3,000 4,000 500 1000 1000 100 Antigen Host Company Catalog # DilutionEvaluation of apoptosis-like cell deathAssay for arginase activityTo PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28250575 determine neural apoptosis-like cell death, terminal deoxynucleotidyl transferase-mediated dUTP end-labeling (TUNEL) staining (In Situ Cell Death Detection Kit, POD; Roche) was performed 48 hours after TBI (n = 5) and the number of TUNEL-positive cells in the ipsilateral hemisphere was then counted and compared gp91 phox-/- with Wt mice in a similar cortical region (40 ?magnification). Production of O2- was determined by in situ detection of oxidized hydroethidium (HEt) [19]. With the animal placed under anesthesia, the HEt solution was administered (1 mg/mL 0.9 NaCl with 1 DMSO) into the jugular vein (n = 3 per group) 48 hours after TBI. Fifteen minutes later, the brain was removed and frozen in GW9662 site blocks and cryosectioned (8 m) in the coronal plane. To demonstrate the cellular distribution of Et, the sections were co-stained with antibodies raised against gp91phox, CD11b, GFAP, or NeuN. Fluorescence was detected using confocal laser microscopy (AX-10, Zeiss, Germany)NO and TNFa measurement in media In situ detection of O2-Arginase is a marker for alternatively activated microglia [20]. Arginase activity was measured according to a previous paper [21] with minor modification. In brief, the cell homogenate was mixed with equal volumes of prewarmed 50 mM Tris-HCl, pH 7.5 containing 10 mM MnCl2 and incubated for 15 minutes at 55 . The mixture was inc.