Sirius red collagen detection kit

Cryosections from the liver (10 μm) were fixed, treated with 3% H₂O₂, incubated with blocking solution (diluted goat serum), and stained with anti-αSMA, TG1, and TG2 antibodies. Staining signals were enhanced using a VECTASTAIN ABC kit and developed with ImmPACT DAB (Vector Laboratories, Burlingame, CA, USA). As a negative control, the primary antibody was replaced with the same amount of rabbit non-immune IgG (NI-IgG) (Sigma; St. Louis, MO, USA). Sections were also stained with hematoxylin and eosin (Leica Microsystems). Collagen was stained using a Sirius Red Collagen Detection Kit (Chondrex, Redmond, USA). Each experiment was conducted in triplicate.

The total collagen content was quantified using the Sirius Red Collagen Detection kit (Chondrex Inc., Redmond, WA, USA). The cell culture medium was collected and mixed with the concentration reagent. The mixture was vortexed and incubated at 4 °C for 24 h. After incubation, the mixture was centrifuged at 10,000 rpm, and the supernatant was discarded. Following this, acetic acid was added to the microtube to dissolve the pellet, and the obtained solution was used as the test sample. Sirius Red solution, for collagen staining, was added to each tube, vortexed, incubated for 20 min at room temperature, and centrifuged. The supernatant was removed by carefully pipetting without disturbing the pellet. Washing solution was added to each tube, and the above steps were repeated once. Finally, an extraction buffer was added and the absorbance was measured at 540 nm using a microplate reader (Sunrise, Tecan, Salzburg, Austria).

Mouse NIH 3T3 fibroblasts (American Type Culture Collection) were incubated at 37 °C under 5% CO₂/95% air. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum (FBS). The concentration of FBS was reduced to 1% at 24 h or 72 h before testing. Some groups of cells were cultured with ramipril (Sigma-Aldrich, St. Louis, MO, USA) and/or TGF-β1 (to simulate the traumatic stimulus). Cell proliferation was detected with the Cell Counting Kit-8 (CCK-8; Dojindo, Japan). Total collagen was detected by the Sirius Red Collagen Detection Kit (Chondrex Inc., Washington, USA). After reading the Manual carefully, we performed our test according to the instructions and procedures.

Protein concentration of mouse urine was measured using bicinchoninic acid assay (BCA) reagents (Pierce). Urinary creatinine was measured using a colorimetric kit (Enzo Life Sciences). Masson's Trichrome or H&E was used to stain five μm histological sections of bladder tissue (performed by WaxIt Histology, Inc.). The Sirius Red Collagen Detection Kit (Chondrex) was used to quantitate collagen in bladder tissue from mice that were 3–3.5 months old. Scoring of the degree of detrusor smooth muscle layering: We estimated the degree of layering by roughly categorizing randomly photographed H&E-stained bladder sections. A score of 0 was given to a normal, compact appearance. Mild to medium layering with some white spaces between smooth muscle layers was scored as 1, and severe layering with a “bacon-like” appearance was scored 2 (S1A Fig).

The NIH/3 T3 mouse fibroblast cell line was purchased from the Cell Engineering Division of the RIKEN BioResource Center (Tsukuba, Ibaraki, Japan). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing antibiotics and 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator containing 5% CO₂. For all the experiments, the cells were used within 3 to 7 passages after the reception. For in vitro functional analysis of CCL8, NIH/3 T3 cells were seeded in a 96-well plate at a density of 1 × 10⁵ cells/mL. Some groups of cells were cultured with varying concentrations of recombinant mouse CCL8 (BioLegend, San Diego, CA) for 24 h. The concentration of FBS was reduced to 1% at 24 h before testing. Total collagen was detected by the Sirius Red Collagen Detection Kit (Chondrex, Woodinville, WA) according to the manufacturer’s instructions.