Direct red 80

Formalin-fixed liver samples were paraffin embedded, sectioned, and stained with hematoxylin and eosin (H&E) or Gömöri trichrome at the Connecticut Veterinary Medical Diagnostic Laboratory (CVMLD), the University of Connecticut. Also, paraffin-embedded liver sections were subjected to Picrosirius red staining. Slides were first deparaffinized and rehydrated by immersion in xylene twice for 5 min, followed by 100% ethanol twice for 5 min. The slides were then immersed through graded ethanol (95%, 70%, 50%) for 5 min each and finally in water for 5 min. Subsequently, the slides were stained with Weigert’s iron hematoxylin (Electron Microscopy Sciences, Hatfield, PA) for 8 min and then washed with water for 10 min, after which they were stained with picrosirius red staining solution (0.1% (w/v) Direct red 80 (MilliporeSigma) in saturated aqueous picric acid (Ricca Chemicals, Arlington, TX)) for 2 h. Slides were then washed with acidified water for 1 min and then twice with 100% ethanol for 5 min. After washing the slides with xylene for 5 min, the stained sections were covered with a coverslip using a DPX mountant (MilliporeSigma), air-dried, and imaged. Picrosirius red positive area was then calculated using Image J image analysis software. All stained images were captured with a 10X objective using the Zeiss Axio Vert A1 (Oberkochen, Germany) equipped with an MRc camera.

The acid -soluble collagen content from the different adipose depots were measured using a sirius red dye binding assay following the protocol described before (Coentro, Capella-Monsonis, Graceffa et al., 2017 (link)). Briefly, the acid-soluble collagen was extracted from 100 mg adipose tissues by homogenization and incubation with 0.5 M acetic acid for 48 h at 4o C. The soluble collagen was obtained by centrifugation and incubated with 1 mL 0.1% Direct Red 80 (Millipore Sigma) in saturated picric acid dye reagent solution for 1 h at room temperature. The collagen–dye complex was precipitated by centrifugation at 10,000 g for 10 min, and the supernatant was removed. The resulting pellets were dissolved in 1 mL 0.5 M sodium hydroxide, and the relative absorbance was measured in a 96-well plate at 540 nm using a microplate reader (Molecular Devices SpectraMax, San Jose, CA). Collagen content was normalized to total protein content determined as described before (Puttabyatappa et al., 2017 ). The inter and intra-assay coefficients of variance for Sircol assay was 1.43 ± 0.32 and 1.71 ± 0.94. The measurable range for this assay is 31.25 to 500 μg/mL.

Sodium hypochlorite solution (concentration: available chloride 10–15%, 425044), acetic acid (concentration≥99.7%, 320099), Direct red 80 (Sirius red, powder, dye content 25%, 365548), ferric chloride (powder, F-7134), picric acid solution (1.3% in H₂O, saturated, P6744) and hematoxylin (powder, H9627) were purchased from Sigma-Aldrich (MO, USA). Ethylenediaminetetraacetic acid (powder, 99%, A10713) was obtained from Alfa Aesar (MA, USA). Paraffin (melting point 56–57°C, 22900700) was acquired from Fisher Scientific (PA,USA). Ethyl alcohol (absolute, anhydrous, 111000200) was purchased from Pharmco (USA), and hydrochloric acid (concentration 36.5–38%) was obtained from VWR Chemicals BDH (USA).