Pathologic findings were graded according to a 5-point semi-quantitative severity-based scoring system as: 0 = normal lung parenchyma, 1 = changes in 1–25%, 2 = changes in 26–50%, 3 = changes in 51–75%, and 4 = changes in 76–100% of examined tissue (Araújo et al., 2010 and Chao et al., 2010). For recipients of GFP marrow transplants, frozen sections were treated with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI)-supplemented mounting medium (Vectashield, Vector Labs, Burlingame, CA), cover slipped and examined for GFP expression by confocal microscopy. Background autofluorescence click here was determined through examination
of 10 simultaneously prepared negative control sections that were stained with DAPI alone. Images were obtained Fluorouracil nmr using a Zeiss LSM-410 laser-scanning confocal microscope (Carl Zeiss Canada Ltd., Toronto, ON, Canada) equipped with GFP (green) and DAPI (blue) filter sets. The number of GFP+ cells per tissue area was determined by the point-counting technique (Weibel, 1990, Araújo et al., 2010 and Abreu et al., 2011) across 10 random, non-coincident microscopic fields. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining was used in a blinded fashion by two pathologists to assay cellular apoptosis (Oliveira et al., 2009). Ten fields per section from the regions with cell apoptosis were examined at a magnification of 400×. A 5-point semi-quantitative
severity-based scoring system was used to assess the degree of apoptosis, graded as: 0 = normal lung parenchyma; 1 = 1–25%, 2 = 26–50%, 3 = 51–75%, and 4 = 76–100% of examined tissue. Quantitative real-time reverse transcription (RT) polymerase chain reaction (PCR) was performed to measure the relative levels of mRNA expression of vascular interleukin (IL)-1β, IL-6, IL-10, caspase-3, vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β, platelet derived growth
DAPT research buy factor (PDGF), and hepatocyte growth factor (HGF). Central slices of left lung were cut, collected in cryotubes, quick-frozen by immersion in liquid nitrogen and stored at −80 °C. Total RNA was extracted from the frozen tissues, using Trizol® reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations. RNA concentration was measured by spectrophotometry in Nanodrop® ND-1000. First-strand cDNA was synthesized from total RNA using M-MLV Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA). PCR primers for target gene were purchased (Invitrogen, Carlsbad, CA). Relative mRNA levels were measured with a SYBR green detection system using ABI 7500 Real-Time PCR (Applied Biosystems, Foster City, CA). All samples were measured in triplicate. The relative expression of each gene was calculated as a ratio of studied gene to control gene (acidic ribosomal phosphoprotein P0 [36B4]) and expressed as fold change relative to Sham-SAL. The following PCR primers were used: PDGF (NM_008808.