Fast green

Six kernels from each ear replicate were fixed and dehydrated using the protocol modified from Livingston et al. (2009 (link), 2013 (link)) and Shu et al. (2015) (link). Briefly, we used a modified FAA fixative consisting of 45% methanol, 10% formaldehyde and 5% glacial acetic acid. The paraffin blocks were sectioned with a RM2255 microtome (Leica) and mounted on slides (Gold Seal). Slides were dried on a hot plate overnight and stored at room temperature. Paraffin was removed by dipping the slides in 100% xylene. Sections were then rehydrated with an ethanol series. Safranin and fast green staining were applied to differentiate tissue structure of maize kernels and the fungus grown in the kernel (Figures 1c–h). The rehydrated sections were stained with safranin, dehydrated with an ethanol series, and counter stained with fast green (Fisher) (Shu et al., 2015 (link)). Stained sections were mounted in permount mounting medium (Fisher) and covered with coverslips. Images of stained tissues were collected on an Eclipse E600 light microscope (Nikon). Images were captured on an Infinity1-3C digital camera, and analyzed with the software Infinity Analyze (Lumenera).

Tubulointerstitial and myocardial collagen deposition was evaluated in Picrosirius-red (PSR)-stained sections. After deparaffinization and rehydration, sections were first counterstained during 15′ with 2.5 mg/mL Fast Green (11443054, Fisher Bioreagents, Altrincham, UK) and diluted in 1% acetic acid. Afterwards, they were washed and stained during 1 h with 0.1% Sirius Red (F3B) C.I.35782 (365548, MilliporeSigma, Darmstadt, Germany) and diluted in saturated picric acid solution (P6744, MilliporeSigma, Darmstadt, Germany). Ten different representative LV microphotographs were obtained at 400× magnification with an Olympus BX61 microscope. Fibrosis was quantified by two independent blinded observers as the percentage of collagen deposition with an automated color recognition processing plugin from the ImageJ analysis software (v1.53a, National Institutes of Health, Bethesda, MD, USA). Perivascular and endocardial collagen was excluded from heart measurements.

Cartilage degeneration was visualized in the tibial articular surface of the tibiotalar compartment using the Safranin O (Sigma‐Aldrich) and Fast Green (Fisher Scientific, Waltham, MA) staining method. A hematoxylin and eosin (Sigma‐Aldrich) staining was also used to better visualize chondrocytes. The timeline of cartilage degeneration was quantified by measuring the thickness of cartilage matrix stained by Safranin O with lacunae containing chondrocytes. Using the ImageJ tracing tool, we traced a line over the thickest region of safranin-stained chondrocytes, perpendicular to the surface of the tibia (4 images per animal, n = 6).