Saturated aqueous picric acid

The type of new bone growth was determined with picrosirius red staining and polarized light microscopy (Rich & Whittaker 2005; Shapiro 2008). Paraffin sections were deparaffinized with xylene and rehydrated through a graded series of ethanols. Slides were stained with picrosirius red (0.5 g sirius red F3B in 500 mL saturated aqueous picric acid, Sigma‐Aldrich) for 1 h and washed briefly in acidified water (5 mL glacial acetic acid in 1 L of water). Slides were dehydrated and mounted with Permount mounting medium (Fisher Scientific). Polarized light microscopy was carried out on an Olympus BX60 upright microscope equipped with filters to provide circularly polarized illumination according to published methods (Rich & Whittaker 2005).

For Picrosirius Red/Fast Green staining, sections were postfixed for 30 min in 10% formalin (Sigma), followed by overnight fixation in Bouin’s solution (Sigma), and then extensively washed with tap water. Sections were incubated for 1 h in Picrosirius Red and Fast Green solution: 0.1% direct red 80 (Sigma) and 0.1% fast green FCF (Sigma) in saturated aqueous picric acid (1.2% picric acid in water; Sigma). Sections were quickly washed ten times in distilled water, twice in 70% ethanol, twice in 90% ethanol, then in 100% ethanol (2 × 15 min), and finally in Safesolv solution (Q Path®, VWR International; 2 × 15 min), before being mounted in safemount medium (Q Path®, VWR International). For Hematoxylin and Eosin staining, sections were incubated for 5 min in Hematoxylin solution, washed ten times for 1 min in running tap water, and incubated for 3 min in Eosin solution. Sections were washed three times in distilled water, twice in 70% ethanol, twice in 90% ethanol, then in 100% ethanol (2 × 15 min), and finally in Safesolv solution (Q Path®, VWR International; 2 × 15 min), before being mounted in safemount medium (Q Path®, VWR International). For AFOG staining, an AFOG kit (100 T, Clinisciences) was used, according to the manufacturer’s instructions, before being mounted in safemount medium (Q Path®, VWR International).

The middle layer of the left ventricular sections (5 µm) was hydrated with Sirius Red (0.5% in saturated aqueous picric acid (Sigma, Kawasaki-shi, Japan) and viewed under polarized light. Ten fields in each region of the heart were randomly selected with three sections in non-consecutive series, and collagen content was quantified as Sirius Red positive areas by use of a Zeiss Axiovert S100 TV Microscope (Zeiss, Jena, Germany) 2.0 (UTHSCSA, San Antonio, TX, USA).

Sirius red is a polyazo dye which is specifically used for staining collagen. The stain dyes collagen bright red, leaving muscle fibers, cytoplasm, and red blood cells a lighter yellow/red color. Picrosirius red differs from sirius red staining with the addition of picric acid which prevents the indiscriminate staining of noncollagenous structures by sirius red.
Sections were fixed in neutral buffered formalin for 30 minutes. Slides were rinsed in dH₂O and stained with Weigert's hematoxylin for 8 minutes. Sections were washed in 3 changes of water followed by staining with picrosirius solution for one hour. Picrosirius solution was made up of 0.5 g sirius red F3B (CI35780, Sigma-Aldrich, USA) in 500 ml saturated aqueous picric acid (197378, Sigma-Aldrich, USA). Sections were washed in 0.5% acetic acid (A6283, Sigma-Aldrich, USA) in dH₂O twice for 5 minutes each. Sections were then dehydrated in changes of ethanol (70%, 95%, and 100%) and then cleared in xylene (296325, Sigma-Aldrich, USA), and cover slips were mounted with mounting media (DPX, 06522, Sigma-Aldrich, USA).