Es were collected on day 28 from allogeneic and syngeneic BMT rats and non-BMT handle rats to examine liver function, also as renal function, using an autoanalyzer. Urine was also collected on day 28, to examine proteinuria and urinary N-acetyl-b-D-glucosaminidase levels. three / 18 Acute GVHD from the order TPPU kidney a,b Target Ab MHC class I T cell CD4+ T cell CD4+ T cell T cell CD8+ T cell White blood cell T cell CD8+ T cell Macrophage Monocyte, Macrophage Fcc receptor III/II IgG1 IgM IgG C3 MHC class II Clone C3 1F4 10B5 W3/25 OX-52 G28 OX-1 Rabbit, Polyclonal OX-8 ED1 HIS48 2.4G2 Goat, Polyclonal Goat, Polyclonal Rabbit, Polyclonal Goat, Polyclonal OX-6 Conjugate FITC FITC Purified APC-Cy7 FITC PE PE-Cy7 Purified Purified Purified Biotin PE-Cy7 TRITC FITC FITC FITC PE Company BioLegend, San Diego, CA BioLegend, San Diego, CA Abcam, Tokyo, Japan BioLegend, San Diego, CA BD Pharmingen, San Diego, CA BioLegend, San Diego, CA BioLegend, San Diego, CA DAKO, Glostrup, Denmark BioLegend, San Diego, CA BMA BIOMEDICALS, Augst, 
Switzerland eBioscience, San Diego, CA BioLegend, San Diego, CA SouthernBiotech, Birmingham, AL MP Biomedicals, Tokyo, Japan MBL, Nagoya, Japan MP Biomedicals, Tokyo, Japan BioLegend, San Diego, CA Anti-rat CD3 Ab Anti-rat CD4 Ab Anti-rat CD4 Ab Anti-rat CD6 Ab Anti-rat CD8a Ab Anti-rat CD45 Ab Anti-human CD3 Ab Anti-rat CD8a Ab Anti-rat CD68 Ab Anti-rat Granulocyte Marker Ab Streptavidin Anti-mouse CD16/32 Ab Anti-mouse IgG1 Ab Anti-rat IgM Ab Anti-rat IgG Ab Anti-rat C3 Ab Anti-rat RT1B Ab Culture sup ATCC, Manassas, VA Assessment: Flow: Flow cytometory; IHC: Immunohistochemistry; IF: Immunofluorescence. American Type Culture Collection. Tetramethylrhodamine isothiocyanate. doi:ten.1371/journal.pone.0115399.t001 Pathology and Immunohistochemistry To study the histological characteristics of GVHD, the skin, liver, intestine, and kidney tissues were fixed in 20 buffered formalin and embedded in paraffin for light microscopic examination. Tissues had been stained with hematoxylin and eosin and periodic and acid-Schiff staining for histopathological examination, and naphthol AS-D chloroacetate esterase staining to detect neutrophils. The principal antibodies used for immunohistochemistry are indicated in four / 18 Acute GVHD of the Kidney detect the donor variety of leukocytes inside the kidney, double stain with fluorescein isothiocyanate -conjugated anti-rat RT1Aa,b D8-MMAF (hydrochloride) web antibody and phycoerythrin -conjugated anti-rat CD45 antibody was performed. To detect CD8+ T-cells or CD4+ T-cells, double stain with FITC-conjugated anti-rat CD3 antibody and PE-conjugated anti-rat CD8a antibody or anti-rat CD4 antibody was performed. To evaluate the expression of MHC class II in renal tubules, immunostaining with PE-conjugated anti-rat PubMed ID:http://jpet.aspetjournals.org/content/123/3/180 RT1B antibody was performed. In every kidney sample, more than 100 cross-sections of renal tubules have been graded semiquantitatively according to the specimens stained for rat RT1B, utilizing the following grading program: absence of MHC class II staining; 0, mild increase of MHC class II staining; 1, moderate boost of MHC class II staining; two, marked enhance of MHC class II staining; three. Real-time Reverse Transcription-Polymerase Chain Reaction for Cytokines Real-time reverse transcription-polymerase chain reaction was performed as described previously, to examine the mRNA expression levels of interferon -c, tumor necrosis issue -a, interleukin -4, and IL-17 in the kidney. Total renal RNA was extracted employing the ISOGEN in line with the manufactur.Es were collected on day 28 from allogeneic and syngeneic BMT rats and non-BMT control rats to examine liver function, too as renal function, working with an autoanalyzer. Urine was also collected on day 28, to examine proteinuria and urinary N-acetyl-b-D-glucosaminidase levels. 3 / 18 Acute GVHD in the Kidney a,b Target Ab MHC class I T cell CD4+ T cell CD4+ T cell T cell CD8+ T cell White blood cell T cell CD8+ T cell Macrophage Monocyte, Macrophage Fcc receptor III/II IgG1 IgM IgG C3 MHC class II Clone C3 1F4 10B5 W3/25 OX-52 G28 OX-1 Rabbit, Polyclonal OX-8 ED1 HIS48 2.4G2 Goat, Polyclonal Goat, Polyclonal Rabbit, Polyclonal Goat, Polyclonal OX-6 Conjugate FITC FITC Purified APC-Cy7 FITC PE PE-Cy7 Purified Purified Purified Biotin PE-Cy7 TRITC FITC FITC FITC PE Enterprise BioLegend, San Diego, CA BioLegend, San Diego, CA Abcam, Tokyo, Japan BioLegend, San Diego, CA BD Pharmingen, San Diego, CA BioLegend, San Diego, CA BioLegend, San Diego, CA DAKO, Glostrup, Denmark BioLegend, San Diego, CA BMA BIOMEDICALS, Augst, Switzerland eBioscience, San Diego, CA BioLegend, San Diego, CA SouthernBiotech, Birmingham, AL MP Biomedicals, Tokyo, Japan MBL, Nagoya, Japan MP Biomedicals, Tokyo, Japan BioLegend, San Diego, CA Anti-rat CD3 Ab Anti-rat CD4 Ab Anti-rat CD4 Ab Anti-rat CD6 Ab Anti-rat CD8a Ab Anti-rat CD45 Ab Anti-human CD3 Ab Anti-rat CD8a Ab Anti-rat CD68 Ab Anti-rat Granulocyte Marker Ab Streptavidin Anti-mouse CD16/32 Ab Anti-mouse IgG1 Ab Anti-rat IgM Ab Anti-rat IgG Ab Anti-rat C3 Ab Anti-rat RT1B Ab Culture sup ATCC, Manassas, VA Assessment: Flow: Flow cytometory; IHC: Immunohistochemistry; IF: Immunofluorescence. American Sort Culture Collection. Tetramethylrhodamine isothiocyanate. doi:ten.1371/journal.pone.0115399.t001 Pathology and Immunohistochemistry To study the histological capabilities of GVHD, the skin, liver, intestine, and kidney tissues had been fixed in 20 buffered formalin and embedded in paraffin for light microscopic examination. Tissues were stained with hematoxylin and eosin and periodic and acid-Schiff staining for histopathological examination, and naphthol AS-D chloroacetate esterase staining to detect neutrophils. The main antibodies used for immunohistochemistry are indicated in 4 / 18 Acute GVHD on the Kidney detect the donor type of leukocytes within the kidney, double stain with fluorescein isothiocyanate -conjugated anti-rat RT1Aa,b antibody and phycoerythrin -conjugated anti-rat CD45 antibody was performed. To detect CD8+ T-cells or CD4+ T-cells, double stain with FITC-conjugated anti-rat CD3 antibody and PE-conjugated anti-rat CD8a antibody or anti-rat CD4 antibody was performed. To evaluate the expression of MHC class II in renal tubules, immunostaining with PE-conjugated anti-rat PubMed ID:http://jpet.aspetjournals.org/content/123/3/180 RT1B antibody was performed. In every kidney sample, far more than one hundred cross-sections of renal tubules have been graded semiquantitatively according to the specimens stained for rat RT1B, applying the following grading program: absence of MHC class II staining; 0, mild enhance of MHC class II staining; 1, moderate raise of MHC class II staining; 2, marked boost of MHC class II staining; three. Real-time Reverse Transcription-Polymerase 
Chain Reaction for Cytokines Real-time reverse transcription-polymerase chain reaction was performed as described previously, to examine the mRNA expression levels of interferon -c, tumor necrosis element -a, interleukin -4, and IL-17 inside the kidney. Total renal RNA was extracted utilizing the ISOGEN based on the manufactur.
