Paraformaldehyde (pfa)

To show the effect of compression on IVDs, safranin O/fast green staining was used. Tissues for histology and immunostaining were fixed in 4% (w/v) paraformaldehyde (Absin, China) for 24 ​h before decalcification in 10% (w/v) ethylene diamine tetraacetic acid (EDTA) (Solarbio, China) solution. Subsequently, the specimens were embedded in paraffin and sliced (7 ​μm) for further safranin O/fast green staining or immunostaining. The sections were then deparaffinized and stained by hematoxylin (Sigma–Aldrich, USA) for 20 ​min, followed by fast green (Sigma–Aldrich, USA) staining for 8 ​min, acetic acid washing for 1 ​s, and safranin O (Sigma–Aldrich, USA) staining for 8 ​min subsequently. Finally, the slides were mounted with neutral resin (Sigma–Aldrich, USA) and scanned before observation.

Organoids were completely harvested using Cell Recovery Solution (Corning, USA). (1) IHC staining: organoids were washed once with PBS 1 ×, dehydrated in 70% ethanol (Sango Biotech, China), and stained in 0.5% eosin (Sango Biotech, China; dissolved in 96% ethanol) for 30 min. After rehydration in 100% ethanol, the organoids were embedded in paraffin. The subsequent procedures were the same as the normal IHC steps. The data were analyzed using ImageJ (https://imagej.net/Download) (2) IF staining: after fixation with 4% (w/v) paraformaldehyde (Absin, China) at 4 °C, the organoids were washed in 0.1% (v/v) Tween-PBS (Sango Biotech, China) and blocked with TritonX-100-BSA solution. Primary antibodies were used to incubate the organoids in 24-well plates at 4 °C overnight. After one wash, the organoids were incubated with corresponding secondary antibodies, before finally being transferred to slides with coverslips and observed under laser scanning confocal fluorescence microscopy (LSCFM) (Leica, FV3000). The detailed procedures were described previously [63 (link),67 ].
Immunostaining of mouse liver tissue slides for markers, including cytokeratin 19 (CK19/Krt19), albumin (Alb), α-SMA, and IL-17A, was performed using a Novo-Light Multiplex Fluorescence Immunohistochemical Kit (WiSee Bio, China). The complete list of immunostaining antibodies used can be found in Table S1.