Dsvi1 brightfield camera

Sections (4μm) of formalin-fixed, paraffin-embedded tissues were deparaffinized with xylene and rehydrated. Serial sections were stained with H&E or Alcian Blue as described [17 (link)]. Immunohistochemistry was performed on a Leica BOND autostainer (Wetzlar, Germany). Slides were scanned in brightfield using the Aperio Digital Pathology System (Leica). Images were analyzed using the Tissue Classifier and Area Quantification modules of the HALO Image Analysis System v.3.0 (Indica labs, Albuquerque, NM). For immunofluorescence antigen retrieval was performed in a pressure cooker using 10mM Sodium citrate, pH 6.0. Primary antibodies were incubated overnight followed by incubation with secondary antibodies and TrueVIEW (SP-8400, Vector Laboratories, Burlingame, CA). Images were captured with a Nikon DSVi1 brightfield camera using NIS Elements 3.2 Basic Research Image software (Nikon Instruments Inc., Melville, NY).

Four-micron sections of formalin-fixed paraffin-embedded tissues were deparaffinized with xylene and rehydrated sequentially. Antigen retrieval was performed in a pressure cooker in Trilogy pH 8.0 Buffer (Cell Marque, Rocklin, CA) and subsequent incubations performed on a Dako Autostainer Plus (Agilent Technologies, Santa Clara, CA). Slides were treated with 3% hydrogen peroxide, blocked with TCT Buffer (0.05M Tris, 0.15M NaCl, 0.25% Casein, 0.1% Tween 20, pH 7.6) and incubated with anti-TNC antibody (1:75) (Novus Biologicals, Littleton, CO) or a matched concentration of rabbit IgG. Poly-HRP anti-Rabbit IgG Polymer (Leica Microsystems, Buffalo Grove, IL) was then applied followed by DAB+ substrate-chromagen (Agilent Technologies, Santa Clara, CA). Slides were counterstained with hematoxylin (Agilent). Serial sections were stained with hematoxylin and eosin for histological analyses. All images were captured with a Nikon DS-Vi1 brightfield camera using NIS Elements 3.2 Basic Research Image software (Nikon Instruments Inc., Melville, NY).