Vessel Recovery in Scleroderma . ... Table 8. Antibodies used for immunohistochemical analysis of skin biopsies. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... Antibody Manufacturer Clone Pretreatment Dilution Secondary/Kit . ... . Signal trasducer and activator of transcription 1 (STAT1){ CellSigTech Tyr701, 58D6 Citrate/heat 1:200 Biomouse/rabbit Elite ... P Selectin (CD62P){ Abcam AK-6 Citrate/heat 1:500 Biomouse/rabbit Elite . ... P Selectin glycoprotein ligand 1 (PSGL1) { Abcam 3E2.25.5 PL1 Citrate/heat 1:200 Biomouse/rabbit Elite . ... Interleukin-1alpha (IL-1a) { R&D Systems Lot #AAB01 Citrate/heat 5 ug/mL Biomouse/rabbit/goat Elite .... Vascular endothelial (VE) Cadherin{ Novocastra BV6 Citrate/heat 1:100 Vector Biomouse Elite ... Platelet endothelial cell adhesion molecule 1 (CD31){ Novocastra 1A10 EDTA 1:400 Mouse/Envision . ... Ulex europaeus lectin{ Vector pronase 1:200 Elite SA (no secondary) .... Interleukin 3 receptor subunit alpha (CD123) { DAKO 7G3 PXXIV 1:250 Mouse/Envision ... smooth muscle actin{ DAKO 1A4 pronase 1:250 Mouse/Envision . ... Protease activated receptor 2 (PAR2){ Zymed sam11 none 1:500 Mouse/Envision ... . Intercellular adhesion molecule 1 (ICAM1){ Chemicon Wcam1 Citrate/heat 1:200 Mouse/Envision ... Vascular cell adhesion molecule 1 (VCAM1){ R&D Systems BBA19 EDTA 1:1000 Vector BioGoat/Elite . ... Ki67 antigen (Ki67){ DAKO MIB-1 Citrate/heat 1:200 Mouse/Enviision . ... Cleaved caspase 3{ Cell Sig Tech #9661 EDTA 1:200 Vector BioGoat/Elite .... Von Willebrand factor{ DAKO M0616 pronase 1:100 Mouse/Envision . . .. Detection for all IHC performed using 3,39-diaminobenzadine (DAB). ... {In Lab IHC .. {Phenopath IHC .. . doi:10.1371/journal.pone.0001452.t008 endothelial (VE) cadherin, alkaline phosphatase and Ulex europaeus lectin. Serial biopsies were stained with all 5 markers and vessels (identified as clusters of cells positive for CD31) were compared. If vessels were found that had CD31 stain but lacked another marker in a serial section, the biopsy was scored as negative for that marker. The observed negative and positive were recorded in 262 frequency tables. RGS5 Stain score Normal biopsies hybridized with RGS5 probes showed 0-1 perivascular cells per high power field. Slides that had more than 2 positive cells per high power field were considered positive. Positive slides always had increased perivascular expression, and some also had many cells positive in the dermal matrix. Negative and positive were recorded in 262 frequency tables. Interleukin-3 receptor alpha subunit (CD123) (high endothelial venule phenotype), smooth muscle actin and P selectin (CD62P) stain score Biopsies were examined for presence or absence of lumens of blood vessels with endothelial cells labeling with CD123, smooth muscle actin and CD62P. Biopsies were scanned until positive vessels were located or until entire biopsy had been examined. Biopsies with positive vessels were recorded as positive. Negative and positive were recorded in 262 frequency tables. Interferon a score Biopsies were scored according to the presence or absence of positive stain anywhere in biopsy. Biopsies with any stain color were scored as positive. Negative and positive were recorded in 262 frequency tables. Vascular cell adhesion molecule 1 (VCAM1), Intercellular adhesion molecule 1 (ICAM1), interleukin-1 alpha (IL-1a), p selectin glycoprotein ligand 1 (PSGL1), protease activated receptor 2 (PAR2), Ki67 antigen (Ki67), CD123 (plasmacytoid dendritic cells), and cleaved caspase 3 stain scores Normal skin biopsies were examined and cells per high power field were quantified. The mean of cells per high power field was calculated and we assumed a normal distribution. We added two standard deviations above the positive range and used that number as a cutoff. Biopsies with an average number of cells higher than the cutoff were scored as positive. Biopsies with an average number of cells lower than the cutoff were scored as negative. Negative and positive were recorded in 262 frequency tables. Signal transducer and activator of transcription 1 (STAT1) stain score Cells with a nuclear stain were considered positive. The numbers of positive cells were counted in 3 high power fields and averaged. The normal skin biopsy range was determined and 2 standard deviations were added to the top of the range for a positive cutoff. Only biopsies with an average number of stained nuclei per high power field greater than the cutoff were considered positive. Negative and positive were recorded in 262 frequency tables. Capillary counts For quantification purposes, we have used site-matched skin biopsies, since capillary loops change in number from site to site. Biopsies of normal, scleroderma and disease controls were stained with CD31. Photographs of entire biopsy to be counted were taken at 206 with an Olympus microscope and Spot camera. Two separate blinded investigators counted the same set of photographs and data are averages of the two counts. ‘‘Profiles’’ are defined as CD31 stained vessels without a central lumen ‘‘Lumens’’ are defined as CD31 stained vessels with central lumens. ‘‘Vessels’’ are defined as the sum of lumens and profiles. Statistics The stain scores for IHC and RNA in situ hybridization were recorded as frequency data and were analyzed for significance using Fishers Exact Significance test. Frequency data were obtained according to above scoring systems and 262 tables were used to record differences between groups. P values were calculated with one tailed probability value. Capillary quantification is a continuous variable and was therefore tested for significance using the students’ t test for independent variables. Values of p,0.05 were considered significant. Standard error was calculated with the standard deviation divided by the sq root of the N. PLoS ONE | www.plosone.org 11 January 2008 | Issue 1 | e1452