Alcian blue solution

Samples were fixed in 4% paraformaldehyde for a day. Cryosections were prepared with a thickness of 10 μm. Air‐dried tissue sections were immersed in tap water to remove the optimal cutting temperature compound (Sakura), stained with Alcian blue solution (Wako, Osaka, Japan) for 3 min, washed with water, stained with hematoxylin (Wako) for 5 min, washed with tap water for several minutes, stained with eosin (Wako) solution for 5 min, and finally washed in 70% ethanol. Sections were then dehydrated with ethanol and mounted using Softmount (Wako).
To visualize the cartilage of the regenerated skeletal elements in whole‐mount preparations, samples were fixed overnight in 10% neutral buffered formalin solution (Wako), incubated in 70% ethanol plus 1% HCl for 3–4 h, and stained with 0.1% Alcian blue in 70% ethanol plus 1% HCl for 2–3 days. Samples were then washed with 4% KOH without agitation for 2 h at room temperature (RT), incubated in a solution of 2% KOH/50% glycerol for 1–3 days, and cleared in 100% glycerol prior to photography.

Dehydrated tissue sections were immersed in tap water to remove Optimum Cutting Temperature (OCT) Compound (Sakura Finetek), stained with Alcian blue solution (Wako) for 3 min, washed with water, stained with hematoxylin (Wako) for 5 min. washed with tap water for several minutes, stained with eosin (Wako) solution for 5 min, and finally washed with 70% ethanol. Sections were then dehydrated with ethanol and mounted using Softmount (Wako, Richmond, VA). PKH-labeled sections were stained with Alcian blue solution for 3 min, washed with water, washed with 70% ethanol, and mounted using Fluoromount (Diagnostic Bio systems).

Whole larvae were fixed in Bouin’s solution overnight. Paraffin sectioning was performed as described in a previous study (31 (link)). Coronal sections (5 μm thick) of larval intestines were stained with hematoxylin and eosin. For Periodic acid-Schiff (PAS) staining, the deparaffinized sections were placed in a 0.5% periodic acid solution (Fujifilm Wako, Osaka, Japan) for 5 min and rinsed twice with distilled water. Sections were stained with Schiff’s reagent (Fujifilm Wako) for 5 min, washed with a sulfurous acid solution (Fujifilm Wako) three times, rinsed under running tap water, and counterstained with hematoxylin. For acidic mucin staining, deparaffinized sections were placed in 3% acetic acid for 3 min and stained with Alcian blue solution (pH 2.5; Fujifilm Wako) for 30 min. The sections were rinsed under running tap water and counterstained with hematoxylin. Images were captured using a DP72 camera (Olympus) under a BX60 microscope (Olympus).