Panoramic scanner

For histological analysis, whole lungs were fixed for 24 h in 4% phosphate-buffered formalin (PFA), dehydrated and embedded in paraffin, and 5 μM sections were stained using a Masson trichrome staining kit (G1340, Solarbio, Beijing, China) following the manufacturer’s protocols. To develop a modified Ashcroft score, 5 fields in each lung section were randomly selected under 100× magnification to obtain the mean score by two pathologists in a blinded fashion [45 (link)]. For immunohistochemical staining of a-SMA and collagen I, tissue slides were dewaxed by dimethyl benzene, polarized with descending concentrations of alcohol, and rinsed with deionized water. Endogenous peroxidase activity was blocked by incubating sections in 3% hydrogen peroxide solution at room temperature for 30 min after antigen retrieval. The 10% normal goat serum was used to block the slides for 1 h. Primary antibodies were incubated overnight at 4 °C followed by incubation of secondary antibody for 60 min at room temperature. The slides were visualized by DAB staining, and counterstaining was performed with hematoxylin. Images of the stained sections were obtained with a panoramic scanner (3DHISTECH, Budapest, Hungar).

The histology of the liver tissues was examined using Oil-O-Red (Abcam, #ab223796, USA) staining techniques. The Oil O Red stains lipid droplets bright red and is routinely used to determine lipid accumulation in the tissues [48 (link)]. The frozen tissues were cut into 5-μm thick sections, fixed with 4% paraformaldehyde at 4 °C for 30 min, and then washed with phosphate buffer saline and 60% isopropanol. The liver sections were stained with Oil Red O stain for 1 h at room temperature and then washed with 60% isopropanol, followed by PBS. The tissue slides were cleared with xylene and were mounted with a coverslip using a DPX mounting medium (histological grade, # 06522, Sigma Aldrich, Saint Louis, MO, USA). The histology of the tissue sections was observed under Nikon Eclipse 80i Microscope (magnification, ×400). The results were analyzed in three randomly selected fields of view in each section using the panoramic scanner (3DHISTECH Ltd., Hungary). The average densities of collagen fibres (Masson’s trichome staining) and fat droplets (Oil O Red staining) were calculated. Oil O Red staining was carried out in the dark.

All mice were sacrificed under anesthesia on day 55. The left rear ankle joint and spleen were collected after treatment. The thymus was weighed to calculate the thymus index by comparing the thymus weight (g) to body weight (g). The ankle joints were collected from indicated mice and fixed in formalin for 24 h, then were subjected to decalcification in 10% EDTA. 4 μm slices of paraffin-embedded joints and spleens were stained for H&E were imaged with a 3D HISTECH panoramic scanner and analyzed with caseviewer software 2.4.0.119028 (3DHISTECH Ltd., Budapest, Hungary). The evaluation criteria for histology images, as well as for the grades of joints and spleens, have been previously reported [26 (link)]. The severity of joint destruction was classified from grade 0 to grade 4 for the intensity of the lining layer hyperplasia, mononuclear cell infiltration, and pannus formation. Five parameters were applied for the pathological evaluation of the spleen: the amount of red pulp, the total number of GCs, cellularity of the periarteriolar lymphoid sheath (PALS), lymphoid follicles, and marginal zone. The inflammation was ranked on a numerical scale from 0–4 (severe change) [26 (link)].