Anti mgp

Immunohistochemistry was performed on a Leica Bond system using the standard protocol F30. The sections were pre-treated using heat mediated antigen retrieval with citrate-based buffer (pH 6, epitope retrieval solution 1) or EDTA based buffer (pH 9, epitope retrieval solution 2) for 20 min. The sections were then incubated with antibody for 30 min at room temperature and detected using an HRP conjugated polymer system in which DAB was used as the chromogen. The sections were counter-stained with haematoxylin and mounted with Aquatex. The following antibodies were used; anti-Pentraxin 3/PTX3 1/500 [MNB1] (Abcam 90806), anti-Cytokeratin 7 1/400 [RCK105] (Abcam 9021), anti-ITGA7 1/500 (Abcam 203254), anti-MGP 1/500 (Abcam 86233), anti-TEM1 1/400 (Abcam 67273), anti-CD31 1/800 [JC70A] (DAKO 20057487), anti-CD68 1/400 (DAKO 20058607), anti-Smooth muscle actin 1/1000 (Abcam 5694), anti-periostin 1/1500 [EPR20806] (Abcam 227049), anti-APOD 1/500 (orb155698), anti-CXCL14 1/1200 (Abcam 137541), anti-Podoplanin 1/750 [D2-40] GTX31231 (lot 821903108).

Cells were harvested and lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor mixture (Roche, Branchburg, NJ, USA). Protein concentrations were measured using BCA protein assay (Pierce, Rockford, IL, USA). The cell lysates were resolved by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. After blocking in 0.1% casein in PBS, membranes were incubated with primary antibodies (anti-MGP, Abcam Cambridge, MA, USA, ab86233, 1:500) and corresponding peroxidase-conjugated secondary antibodies (Jackson Immuno Research, West Grove, PA, USA). Signals were developed using Hyglo chemiluminescent reagent (Invitrogen) and detected using a ChemiDoc MP (Bio-Rad, Hercules, CA, USA).