Sirius red

We deparaffinized and rehydrated liver paraffin sections, then detected collagen content by Sirius red (Polyscience, 214901) staining per the manufacturer’s protocol. For quantitation, we used 15–20 nonoverlapping slides imaged by a whole-slide scanner (Aperio AT2 scanner) with ImageJ (National Institutes of Health).

For the histopathological analysis, the heads of five mice per group were collected. Detailed experimental procedures have been previously described^{46 (link),47 (link)}. Different protocols were used to evaluate the degrees of inflammation, polyp formations, and epithelial disruptions. Hematoxylin and Eosin (H&E) staining was used for the observation of tissue architecture and cellularity; Sirius red (Polysciences Inc., Warrington, PA, USA) staining, for eosinophils; anti-neutrophilic antibody (NIMP-R14; Abcam, Cambridge, UK) staining, for neutrophil; Giemsa (Sigma-Aldrich) staining, for mast cells; and Periodic acid‑Schiff (Sigma-Aldrich) staining, for goblet cells.
The numbers of positive cells were determined in five high-power fields (HPF; 400×) by two independent observers who were blinded to the group assignment. If the examiners had a disagreement, a consensus was reached by reviewing the specimen under a multihead microscope by our research team. NPs were defined as distinct mucosal bulges with neutrophilic infiltration and/or microcavity formation, as previously described^{8 (link)}. The results of inflammatory and secretory cells were expressed as cells per high-power field.

Native and decellularized
brain samples were fixed with 3.7% formaldehyde solution at 4 °C
overnight. Fixed samples were embedded in optimum cutting temperature
solution (OCT, Tissue-Tek), frozen, and 10 μm cryosections were
cut and mounted on glass slides. For DNA staining with Hoechst, slides
were hydrated and stained for 15 min in 1 μg/mL Hoechst solution
(Invitrogen) in PBS and visualized by fluorescence microscopy. For
Haematoxylin & Eosin staining, slides were hydrated and stained
with Mayer’s Haematoxylin for 3 min, followed by a 3 min wash
with tap water. Then, slides were immersed in 95% ethanol and stained
with Eosin alcoholic solution for 45 s. For collagen staining, Sirius
Red (PolySciences) in a saturated aqueous solution of picric acid
was used. Slides were stained for 1 h and then rinsed in 0.5% acetic
acid solution. Alcian blue staining was performed for sGAG assessment.
Slides were hydrated and stained with 1% Alcian Blue in 3% acetic
acid solution at pH 2.5 (Sigma) for 30 min, followed by a 2 min wash
with tap water. Delipidization was examined by Oil Red O staining.
Slides were rinsed with tap water and incubated in 60% isopropanol.
Then, slides were incubated in Oil Red O stain (Sigma) for 15 min
and washed with distilled water. After staining, all slides were dehydrated,
mounted, coverslipped, and visualized by light microscopy.