Synovial tissue samples were frozen in Tissue-Tek OCT medium (Miles, Elkhart, IN) or formalin fixed and paraffin embedded (FFPE).
For immunohistochemistry, antigen retrieval was performed at pH 9 on FFPE sections using Tris-EDTA, 0.05% Tween 20 (10mM Tris Base, 1mM EDTA Solution, 0.05% Tween 20). Sections were stained using anti-FAPα (R&D) and anti-goat Horseradish peroxidase (HRP) (Dako). HRP staining was developed using the ImmPACT DAB Peroxidase HRP Substrate (Vector Labs). Images were acquired using the Zeiss Axio Scan and analysed with Zen lite 2012 software (Zeiss). Number of pixels was quantified and divided by a manually defined tissue area and the average number of pixels per unit area (pixel/UA) was calculated.
For immunofluorescence, acetone fixed frozen sections were incubated with anti-FAPα (F11-24, eBioscience), anti-PDPN (NZ-1.3, eBioscience) and anti-THY1 (Thy-1A1, R&D). These were detected with goat anti-mouse IgG1 FITC, anti-mouse IgG2a TRITC and anti-mouse IgG2b Cy5 (all Southern Biotech). To increase signal from FITC-channel, goat anti-FITC Alexa-488 antibody (Invitrogen) was used. Images were acquired using a Zeiss LSM 510 confocal microscope and ZEN pro 2011 imaging software.
For immunohistochemistry, antigen retrieval was performed at pH 9 on FFPE sections using Tris-EDTA, 0.05% Tween 20 (10mM Tris Base, 1mM EDTA Solution, 0.05% Tween 20). Sections were stained using anti-FAPα (R&D) and anti-goat Horseradish peroxidase (HRP) (Dako). HRP staining was developed using the ImmPACT DAB Peroxidase HRP Substrate (Vector Labs). Images were acquired using the Zeiss Axio Scan and analysed with Zen lite 2012 software (Zeiss). Number of pixels was quantified and divided by a manually defined tissue area and the average number of pixels per unit area (pixel/UA) was calculated.
For immunofluorescence, acetone fixed frozen sections were incubated with anti-FAPα (F11-24, eBioscience), anti-PDPN (NZ-1.3, eBioscience) and anti-THY1 (Thy-1A1, R&D). These were detected with goat anti-mouse IgG1 FITC, anti-mouse IgG2a TRITC and anti-mouse IgG2b Cy5 (all Southern Biotech). To increase signal from FITC-channel, goat anti-FITC Alexa-488 antibody (Invitrogen) was used. Images were acquired using a Zeiss LSM 510 confocal microscope and ZEN pro 2011 imaging software.