2.1. Bleomycin induced pulmonary fibrosis in mice

All animal experiments were carried in accordance with the procedures approved by the Institutional Animal Care and Use committee at the University of Illinois at Chicago, called the Animal Care Committee (Protocol #15-240). The bleomycin-induced rodent model for pulmonary fibrosis was developed as per the review of various animal models by Moore et al., [24]. Mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (5 mg/kg) for bleomycin instillation. The rodent was placed at 45° angle on a platform hanging by its incisors. The tongue was pulled out gently and held to the side using forceps, and 1.5 U/kg of body weight of bleomycin sulfate (Hospira Inc., Lake Forest, IL, USA) was given to the mouse by intratracheal injection with a maximum volume of 50 μL. The mouse was then taken out of the platform and placed under a heating lamp for a few minutes, before returning it to the cage. Based on the time point of harvest post-bleomycin challenge, the animals (N=3 in each group) were grouped as 1. Day 0 (control); 2. Day 7 (inflammation); 3. Day 14 (onset of fibrosis); 4. Day 21 (fibrosis); and 5. Day 28 (recovery phase) [46]. At each time point, the sacrificed animals were euthanized and the lungs were harvested, fixed in formalin, and embedded in paraffin.

Formalin-fixed paraffin embedded (FFPE) lung tissues were sectioned by a microtome at 4 μm thickness onto low-emissivity glass slides (Kevley Technologies, Chesterland, Ohio), suitable for transflectance-mode infrared imaging. FFPE tissues were used for this study, as while there is loss of some biomolecular constituents (such as lipids) during sample preparation, FFPE samples are the most commonly used format in anatomical pathology. In addition, FFPE tissues typically have less sectioning artefacts resulting from sample preparation. Slides were then dewaxed prior to IR imaging using serial washes with xylene [19]. Adjacent tissues sections were cut onto regular glass slides and stained with hematoxylin and eosin (H&E) and Masson’s trichrome by the University of Illinois at Chicago Research Histology and Tissue Imaging Core (RHTIC). The stained sections were later scanned using the Leica Aperio Scanscope CS (Buffalo Grove, IL) and were examined by a pathologist (A.K.B) to identify the key areas in the lung samples and in particular the regions of collagen deposition, based on trichrome staining.