Spot rt3 microscope

OCT-embedded, frozen tumor tissues were cryosectioned in a Leica CM3050 S Cryostat at 10μm thickness for all immunohistochemical (IHC) and immunofluorescence (IF) studies. Slides were incubated overnight in primary antibody at 4°C. Secondary/tertiary antibodies were added and sections were developed and counterstained according to the appropriate protocol. For IHC, the following antibodies were used: anti-human fibronectin (Sigma #F3648), anti-mouse versican (EMD Millipore #AB1033, Burlington, MA, USA), biotinylated hyaluronic acid binding protein, HABP (EMD Millipore #385911), Rabbit IgG isotype control (Santa Cruz Biotechnology #sc-2027, Dallas, TX, USA), and biotin-SP-conjugated goat anti-rabbit IgG secondary antibody (Jackson Immunoresearch #AB2337959, West Grove, PA, USA). The following antibodies were used for IF: anti-human FAP antibody (R&D Systems #AF3715, Minneapolis, MN, USA or Abcam #53066, Cambridge, MA, USA) or sheep IgG isotype control (R&D Systems #5001A), and AF488-conjugated donkey anti-sheep IgG secondary antibody (ThermoFisher #A11015, Waltham, MA, USA). All imaging was completed with the Olympus Spot-RT3 microscope at 20X magnification. For quantification purposes, 3-5 different tumor samples (n≥3) from each experimental group were used. The mean area of positive staining and mean fluorescence intensity were determined using Fiji ImageJ Software.

Non-extracted (containing cells) FDMs were generated (as described in [25 ]) and analyzed for their expression of Fibronectin and Smad7 by immunofluorescence using antibodies against fibronectin (Sigma #F3648), Smad7 (ThermoFisher #42-0400), AF488-conjugated goat-anti-rabbit IgG (ThermoFisher #A11070) or AF594 conjugated anti-rabbit IgG (ThermoFisher #A11072). Imaging was completed with the Olympus Spot-RT3 microscope at 20X magnification. For quantification, 4-5 different fields of view of each type of matrix were used, and the matrix assembly assay was completed at least 3 independent times. The total area of positive FN staining and the % of SMAD7-positive nuclei was determined using ImageJ software. To calculate the % of SMAD7-positive nuclei, the following formula was utilized: (# of SMAD7 particles ÷ # of DAPI-stained nuclei) x 100%.