Shandon varistain 24 4 automatic slide stainer

Control or IPF lung tissues (n = 3 donors) were fixed and embedded in paraffin wax; tissue sections (4 µm) were processed and stained as previously described [20 (link)]. Briefly, the tissue sections were de-waxed, rehydrated and incubated with 3% hydrogen peroxide in methanol for 10 min to block endogenous peroxidase activity. Sections were then blocked with normal goat serum and incubated at room temperature with a primary antibody against ZEB1 (1:500, Sigma), followed by a biotinylated secondary antibody (1:500, Vector Laboratories Ltd., UK); antibody binding was detected using streptavidin-conjugated horse-radish peroxidase and visualised using DAB (DAKO) before counterstaining with Mayer’s Haematoxylin. For H/E stain, Shandon Varistain 24-4 automatic slide stainer (Thermo Fisher Scientific) was used. For tinctorial stain, Trichrome stain (Abcam ab150686) was used according to the manufacturers’ instructions. Images were acquired using an Olympus Dotslide Scanner VS110.

The arteries were gently positioned upright (7-day samples) and horizontal (28-day samples) in heated molten 1.5% (w/v) agar in 10% (v/v) formalin/PBS. The agar plugs were processed using the Shandon Excelsior Tissue Processor (Thermo Electron Corporation). Three to 4 μm transverse sections (7-day samples) and longitudinal sections (28 days samples) were cut and mounted onto Superfrost Plus slides. Serial sections were collected and stained systematically after reaching the point of the open and complete lumen and length of the artery, to ensure comparison of the same region in each artery for the individual analyses. For visualization and measurement of the vessel and lesion, Elastic Van Gieson staining was performed in a Shandon Varistain 24-4 Automatic Slide Stainer (Thermo Scientific) and analyzed using ImageJ software. Immunohistochemistry and immunofluorescence were utilized to detect expression of primary antibodies (Major Resources Table). Nonimmune IgG of the same species as the primary was used as negative control in all protocols at the same concentration as the primary antibody to demonstrate the specificity of the protocol.