4.4. Histochemistry

Histochemistry was performed with 10% buffered formalin-fixed, paraffin-embedded samples. The fixation time was 24 h. Samples were thoroughly deparaffinized with xylene and gradually rehydrated with decreasing concentrations of ethanol with the last step being pure distilled water.

Hematoxylin and Eosin (HE) staining (Nacalai Tesque) was performed as follows: deparaffinized samples were immersed in hematoxylin stain until they reached the desired intensity. Every 10 s the staining was assessed through the microscope. After washing, the slides were immersed in 0.1% eosin stain for 5 min. After a brief prewashing with 50% ethanol, the samples were quickly dehydrated with an increasing ethanol concentration. Thereafter, xylene was used as the final step before mounting.

Mallory staining was performed as recommended by the manufacturer’s protocol (Sigma-Aldrich, Tokyo, Japan), except the incubation time for the Biebrich Scarlet-acid fuchsin and aniline blue solution was increased to 10 min. After staining the samples were dehydrated as described above and mounted. All pictures (white light and GFP) were taken with Biorevo BZ-9000 (Keyence, Tokyo, Japan).