E4009

Rat spinal cords were fixed in 4% paraformaldehyde (16005, Sigma-Aldrich, St. Louis, MO, USA) for 24 h, followed by transparentization using xylene (95682, Sigma-Aldrich, USA), dehydration with gradient ethanol, and embedment in paraffin (1496904, Sigma-Aldrich, USA). The paraffin-embedded spinal cords were sliced into 5 μm thick sections by using a slicer (pfm3005E, Dakewe Biotech Co., Ltd, Shenzhen, China), dewaxed by xylene, and rehydrated by gradient ethanol. Then, the sections were stained with hematoxylin (H3136, Sigma-Aldrich, USA) for 7 min, and later differentiated by 1% hydrochloric alcohol (56694, Sigma-Aldrich, USA) for the removal of excessive pigments. After developing blue in weakly alkaline water, the sections were stained with eosin (E4009, Sigma-Aldrich, USA) for 2 min. Following rinse with distilled water, dehydration using gradient ethanol, and transparentization utilizing xylene, the stained sections were observed by an optical microscope (IX71; Olympus, Tokyo, Japan) under ×100 magnification.

EY staining was performed as described (Heustis et al., 2012 (link)). Briefly, synchronized wildtype adult worms were washed and incubated with 0.15 mg/mL from a 5 mg/mL stock (dissolved in 70% ethanol; Eosin Y; Sigma-Aldrich, E4009) in 500 µL of liquid NGM for 3 hr in the dark. Worms are then prepped for microscopic analysis as described above. Note that Eosin Y stock should be stored at –20°C and before its use it should be incubated at 55°C for ~2 min and vigorously vortexed to ensure its solvation.

Sperm quality was assessed according to sperm motility, sperm count and morphological abnormality. Sperm motility was expressed as the percentage of motile sperm evaluated microscopically under a magnification of 40× in four random microscopic fields, and at least 200 sperm cells were counted. The sperm count was measured using an automated cell counter (TC20, Bio-Rad, Hercules, CA, USA).
For sperm morphology, sperm slides were fixed with methanol (Honeywell, Morris Plains, NJ, USA) and stained with an eosin Y (E4009, Sigma-Aldrich, Saint Louis, MO, USA) and ethanol (Bioman, Taipei City, Taiwan) mixture. Then, the slides were rinsed with 75% ethanol (Bioman) and dried. Finally, the percentage of morphologically normal sperm within at least 100 sperm cells was calculated.

Sperm samples obtained as described previously were immediately assessed in terms of sperm function parameters, including sperm motility, sperm count, and morphological abnormality. Samples from all groups were diluted with PBS, and the motility evaluated under a light microscope (E400, Nikon, Tokyo, Japan). Sperm motility was expressed as the percentage of motile sperm over the total spermatozoa counted. At the same time, samples were incubated at 37 °C for 15 min and then diluted with PBS to estimate the sperm count using an automated cell counter (TC20, Bio-Rad, Woodinville, WA, USA) and the percentage of sperm of normal morphology. A drop of sperm sample was placed on a slide and air-dried at room temperature. The dried sperm sample was then fixed with methanol (Honeywell, Redmond, WA, USA) for 5 min and stained with an eosin Y (E4009, Sigma-Aldrich, St. Louis, MO, USA) and ethanol (Bioman) mixture. After 15 min, the slides were rinsed with 75% ethanol (Bioman) and dried. The slides were assessed using a light microscope (DM1000, Leica, Wetzlar, Germany) and the percentage of sperm of normal morphology in a minimum of 100 spermatozoa was assessed. The rest of the sperm samples were centrifuged at 2000× g for 6 min. After discarding the supernatant, the sperm pellets were collected and stored at –80 °C.

For observing the histopathological changes in nasal mucosa after CGA or Dex intervention, a 4-cm-thick mouse nasal mucosa was fixed in 4% paraformaldehyde (P6148, Sigma–Aldrich, U.S.A.), dehydrated by ethanol (E7023, Sigma–Aldrich, U.S.A.), blocked in paraffin (327204, Sigma–Aldrich, U.S.A.), and sliced. Then, the sliced mouse nasal mucosa was soaked in xylene (95670, Sigma–Aldrich, U.S.A.) for dewaxation. Gradient ethanol 100, 90, 80, and 70% was used to rehydrate mouse nasal mucosa slices. Mouse nasal mucosa slices were stained by Hematoxylin (H3136, Sigma–Aldrich, U.S.A.) for 15 min, differentiated by being soaked in 5% acetic acid (A6283, Sigma–Aldrich, U.S.A.), and immersed in distilled water for developing blue. Next, mouse nasal mucosa slices were stained by Eosin (E4009, Sigma–Aldrich, U.S.A.) for 10 min. Gradient ethanol 70, 80, 90, and 100% was used to dehydrate mouse nasal mucosa slices. After soaking in xylene twice, mouse nasal mucosa slices were sealed by Canada balsam (60610, Sigma–Aldrich, U.S.A.) and observed using an optical microscope (M3800, Fisher Scientific, Waltham, MA, U.S.A.).

Hearts were removed from the chest cavity and weighed on a Mettler Toledo, AT201 analytical scale. The skeletal muscle of the lower hindlimbs was dissected out, and the length of the left tibia was measured. Heart sections, as described above, were stained with haematoxylin (Sigma‐Aldrich, H9627) and eosin (Sigma‐Aldrich, E4009), and was carried out by the Department of Pathology at the Cincinnati Children's Hospital Medical Center. Sections were examined for sarcolemma staining labelled with wheat germ agglutinin (WGA, Invitrogen), according to manufacturer's instruction. Cardiomyocyte cross‐sectional area was measured using ImageJ Software.4, 8