Picro sirius red kit

The level of fibrosis was determined using Sirius red staining. The rats were sacrificed by perfusion with normal saline. The livers were fixed with 4% paraformaldehyde and embedded in paraffin. The 5 μm slides were incubated with the Picro Sirius Red kit (ab150681, Abcam) for 30 min at 25°C to visualize collagen, while the nuclei were stained with hematoxylin. Images were captured using a bright-field microscope (BX61, Olympus, Japan). Quantification of the Sirius red area was performed using ImageJ (version 1.53j).

IVD tissues were fixed in 4% saline-buffered formalin for 48 h. Paraffin sections of 6 μm were cut in the sagittal plane. safranin-O/fast green staining was performed for an overall assessment of the proteoglycan content of the tissue (stained pink/red). Briefly, sections were incubated in Weigert’s iron haematoxylin (Waldeck, Münster, Germany) for 5 min to stain the cell nuclei and, subsequently, immersed in 0.01% fast green (Waldeck) solution for 5 min to stain the collagen. Following rapid rinsing in 1% acetic acid (VWR, Radnor, PA, USA) solution, slides were immersed for 5 min in 0.1% safranin-O (Sigma-Aldrich) solution to detect proteoglycan deposition. The picrosirius red kit (Abcam, Cambridge, UK) was used to stain collagen. Sections were stained with picrosirius red solution for 1 h at room temperature. Birefringent collagen fibres were imaged with polarised light (Axiophot 451887, Zeiss, Jena, Germany). The colour hue corresponds to relative fibre thickness from thin green fibres to increasingly thick yellow, orange and red fibres. All images were captured with the same parameters. Area or red (1–9 nm; 230–255 nm), orange (10–38 nm), yellow (39–51 nm) and green (52–128 nm) fibres were quantified using ImageJ software following Pereira et al. (2016) [60 (link)].

Four to eight mice from each group were randomly selected. Livers were fixed in 4% paraformaldehyde in PBS (Sigma, Steinheim, Germany) at room temperature for 48 h, embedded in paraffin, sectioned at 6 μm, and stained with hematoxylin and eosin (H&E). Liver fibrosis was assessed by staining with a Picro Sirius Red kit (Abcam, Cambridge, MA, USA, #ab150681). For immunohistochemistry, paraffin-embedded sections were stained with antibodies against F4/80 (1:60; AbD Serotec, Oxford, UK, #MCA497GA), α-smooth muscle actin (α-SMA, 1:300, Abcam, #ab7817), ICAM-1 (1:200; Santa Cruz, CA, USA, #sc8439), and CD31 (1:200, R&D System, MN, USA, #AF3628). Images were using an eclipse microscope (Nikon, Tokyo, Japan), and the Sirius Red, F4/80, α-SMA, ICAM-1, and CD31-positive areas were quantified using Image J.