Quick hardening mounting medium

Masson’s trichrome staining was performed with a Masson staining kit (cat #G1340, Solarbio) according to the manufacturer’s instructions. After 10-min fixation in acetone-methanol, the sections were dipped in Weigert iron hematoxylin solution for 5 min. Subsequently, sections were rinsed in tap water for 5 min and incubated in a solution containing 0.04% acidic fuchsia and 0.02% azophloxine for 20 min before rinsing with a 1% acetic acid solution. Next, the sections were placed in a 2.5% phosphotungstic acid solution for 10 min and rinsed again with a 1% acetic acid solution. Finally, sections were dehydrated in ethanol and xylene, placed in a toluene solution, and mounted with a quick-hardening mounting medium (cat #03989, Sigma-Aldrich). The fractional areas of the collagen components (blue) in the mouse aortic valve region were obtained using a DM3000 microscope; ten randomly selected microscopic fields per specimen were analyzed.

Teratogenicity induced by GO was evaluated through the analysis of chironomid mentum deformities following the 7 days of exposure. Such deformities can appear after every larval molt occurring between each development instar in the presence of a teratogenic compound. Thus, only cephalic capsules from 4th instar larvae were considered for teratogenicity assessment to integrate the potential effects occurring after three successive molts.
Sample preparation was performed as follows: cephalic capsules were first discolored with potassium hydroxide (15%) at 95 °C for 12 min prior to potassium hydroxide being replaced by ethanol (70%) for 12 h to stop the reaction. Then, capsules were mounted on a microscope slide and fixed with Quick-hardening mounting medium (Sigma Aldrich, Saint-Quentin-Fallavier, France).
Cephalic capsules were observed under a light microscope, and mentum anomalies were classified according to the work of Warwick and Tisdale [50 (link)]. Blind analysis was performed, and as previously suggested, only anomalies classified as tooth deletions, additions and Khön gaps (not observed during the experiment) or first tooth split were considered to avoid analytical inconsistencies [51 (link)] (Figure 1A–D). To evaluate the severity of the anomalies in affected larvae, a scoring was used based on the previous work from Vermeulen and collaborators [52 (link)].

Colon tissue sections were stained with filtered 2.9% hematoxylin (Sigma-Aldrich, St. Louis, MO, United States). After rinsing in cool running water for 5 min, sections were dipped in 0.5% eosin (Sigma-Aldrich, St. Louis, MO, United States) five times. After rinsing in water for 5 min, sections were sequentially dehydrated using 50% ethanol, 70% ethanol, and 95% ethanol. Sections were then dipped in xylene (Sigma-Aldrich, St. Louis, MO, United States) for cleaning and mounted with quick-hardening mounting medium (Sigma-Aldrich, St. Louis, MO, United States) for observation. The histological evaluation of colon injury was conducted by three investigators in a blind setting. The criteria for assigning histological evaluation scores of colon injury are described in Table 2 (Fung and Putoczki, 2018 (link)).