Zinc formalin

Six‐ to eight‐week‐old C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, Maine). Mouse experiments were approved by the MUSC IACUC. Pulmonary fibrosis was induced in mice following intratracheal administration of 1.2 mU/g bleomycin mixed with 10 µg rhIGFBP‐4 or vehicle in a volume of 50 µL. rhIGFBP‐4 or mutant IGFBP‐4 were administered to different mice on days 0, 3, and 6 post‐bleomycin for a total of three doses. On day 21, mice were euthanized and lungs were gently perfused with 1X PBS. The left lung was collected for hydroxyproline assay. The right lung was further perfused with 10% zinc formalin fixative (Polysciences Inc, Warrington, PA) and fixed in 10% zinc formalin for 48 hours prior to paraffin embedding and histological analysis.

For histological analysis, 0.5 cm distal colon was fixed in Zinc Formalin (Polyscience Inc.) for 3 hours and then embedded in paraffin blocks. We then prepared 5 μm paraffin cross-sections, which were used for hematoxylin and eosin (H&E) staining (Mayer’s Hematoxylin solution, 1% Eosin Solution, Wako) following standard procedures. The histological images were captured using the BX51-P Polarizing microscope (Olympus) and processed with the Olympus D.P. Controller 2002 software.
For immunohistochemistry analysis, 0.5 cm distal colon was fixed overnight in 4% paraformaldehyde (PFA) (Wako) at 4°C and mounted in embedding medium Tissue-Tek O.C.T Compound (Sakura). The tissues were cut into 8 μm sections and permeabilized with 0.2% saponin (Nacalai Tesque) in PBS. The sections were then blocked with 5% goat serum (Wako) for Mucin 2 detection. Subsequently, the sections were stained with anti-Mucin2 (1:200, rabbit, clone: H-300, Santa Cruz Biotechnology) at 4°C overnight. For the second antibody, Alexa Fluor 488 conjugated donkey anti-rabbit IgG antibody (1:400, Thermo Fisher Scientific) was used with DAPI (1:1000, Dojindo). The sections were assessed using the Leica TCS SP8 (Leica Microsystems).