. .. Table 6. Age, sex and anatomic location of skin biopsies from . . normal controls . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... Patient# Age Sex Biopsy Location . .... 1 un un scalp ... 2 un un un . ... 3 un un scalp .... 4 45 F forearm ... 5 un un un . ... 6 un un scalp . ... 7 un un scalp ... 8 un un breast . ... 9 un un abdomen . ... 10 un un breast .... 11 50 F forearm ... 12 46 F forearm . ... 13 51 F forearm .... 14 41 M forearm ... 15 41 F forearm . ... 16 64 F forearm . ... 17 36 M forearm ... 18 40 F forearm . ... 19 59 F arm . ... 20 47 F posterior shoulder .... 21 73 F lateral neck ... 22 37 F arm . ... 23 54 F posterior neck . ... 24 un un thigh . . . . Abbreviations: un-unavailable .. . doi:10.1371/journal.pone.0001452.t006 RNA in situ hybridization The protocol for RNA in situ including construction and amplification of probe was based on the protocol in the Roche DIG application manual[76]. Proteinase K digestion was performed at a concentration of 5 mg/mL for 20 minutes. Probe concentra- tions were 1 ng/uL and hybridizations were performed at 42uC overnight. Post hybridization washes followed this protocol: low stringency 4615 minutes at 37uC, then high stringency 2630 min- utes at 55uC. Detection with alkaline phosphatase was performed with anti DIG Fab (Roche) at a dilution of 1:500 and the chromogen used was Vector Red (Vector Labs). RNA Probe generation Probe for RGS5 were made from plasmids with a 1.7 kb fragment of human RGS5 long previously characterized and published by us[40]. A 1.7 kb segment of DNA contaiing IFNA1 and a 2.2 kb segment for IFNA2 were amplified from from a human Sanger DNA library clone (#RP11-354P17 GenBank accession # AL353732). The plasmids were subcloned using the Stratgene pPCRScriptamp kit. In vitro transcription was performed with DIG RNA labeling kit (Roche) and RNA was isolated (Agilent low RNA input fluorescent linear amplification kit protocol, 2006 version 4, page 19). Standard controls using sense RNA probes were run at the same concentration as the antisense probe with each experiment and showed no specific signal. Positive and negative tissue controls were included in every experiment as well Vessel Recovery in Scleroderma . ... Table 7. Age, sex, disease subtype and duration and anatomic ... site of skin biopsies of scleroderma patients .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... Patient# Age Sex Disease Type Biopsy Location Disease Duration . ... 1a 47 F Diffuse Forearm 1 year . ... 1b 48 ‘‘ 2 years ... 1c 51 ‘‘ 5 years . ... 2a 44 F Diffuse Forearm 1 year . ... 2b 46 ‘‘ 3 years .... 2c ‘‘ Back ‘‘ ... 2d 48 Forearm 5 years . ... 3 47 M Diffuse Forearm 1 year .... 4a 44 F Diffuse Forearm 1 year ... 4b ‘‘ Upper Arm 1 year . ... 5 un M Diffuse un un . ... 6 51 F Diffuse Forearm 1 year .... 7 33 M Diffuse Forearm 1.5 years ... 8 35 M Diffuse Forearm 1&emsp14;year . ... 9 44 M Diffuse Forearm 6 months .... 10 28 F Diffuse Forearm 2.5 years ... 11 71 F Diffuse Forearm 6 months . ... 12 33 F Diffuse Forearm 1.5 years . ... 13 56 F Diffuse Forearm 8 months ... 14 46 F Diffuse Forearm 8 months . ... 15 32 F Diffuse Forearm 10 months . ... 16 44 F Diffuse Forearm 7 months .... 17 47 F Diffuse Forearm 2.5 years ... 18 39 F Diffuse Forearm 6 years . ... 19 55 F Diffuse Forearm 9 months .... 20 40 M Diffuse Forearm 1&emsp14;year ... 21 44 F Diffuse Forearm 6 months . ... 22 45 F Diffuse Forearm 5 years . ... 23 un un Diffuse Upper Arm un ... 24 43 F Diffuse Forearm 15 months . ... 25 37 F Diffuse Upper arm 7 months . ... 26 50 F Diffuse Forearm 1&emsp14;year .... 27 46 F Diffuse Upper arm 11 months ... 28 40 F Diffuse Forearm 2 years . ... 29 55 F Diffuse Forearm 2 years . ... 30 36 M Diffuse Thigh 15 months . . . . Abbreviations un = unavailable at this time .. . doi:10.1371/journal.pone.0001452.t007 to confirm signal. Standard blast searches were done for the DNA segments amplified and no major non-specific cross reactivity was found to other human DNA. Blast searches were done against the other interferon genes. Minor similarity found between interferon a genes and IFNW. No similarity was found between the interferon a genes and IFNB or IFNG. Scoring methodology All high power fields are at a magnification of 206. Endothelial markers Platelet endothelial cell adhesion molecule 1 (CD31) yielded a predictable staining pattern for all three groups of patients, and was used as a control for the other endothelial markers including von Willebrand factor, vascular PLoS ONE | www.plosone.org 10 January 2008 | Issue 1 | e1452