A Comparison of Fixation Methods and Staining Effects of Fish Mucous Cells and Surface Mucus

To compare the fixation effect of four fixation methods on fish mucosal tissues, and screen the fixation methods suitable for the preservation of mucous cells and their secreted mucus, in this study, the mucosa-associated lymphoid tissues (MALTs) of flounder (Paralichthys olivaceus) were fixed by using different protocols including fixation method (Methacarn and Carnoy fixative solution) and fixed time (2 h and 24 h), and paraffin sections were prepared. After hematoxylin-eosin (HE) staining, Alcian blue-periodate Schiff reaction (AB-PAS), and combined lectin- and immuno-histochemical staining (CLIH) were performed, the fixation and staining effects were evaluated based on histological characteristics and three parameters including goblet cell number, mucin staining area and mucus layer integrity. The results showed that AB-PAS staining clearly distinguished neutral mucin, acidic mucin and mixed mucin, and identified 4 types of mucous cells, and exhibited a better staining effect on mucous cells and mucus layer than HE staining. By using different fixation methods and fixed times, tissue sections with relatively complete structure and clear mucous cells were obtained although a certain degree of difference existed in the fixation and staining effect. In Methacarn fixation groups, the tissue morphological integrity, staining properties and mucous layer preservation effect were obviously better than those of Carnoy's fixation treatment. Of the third, Methacarn fixation solution for 2 h was found to be the best fixation protocol. In this fixation group, the mucosal tissue morphology was more complete and clearer, with less mucus shedding and higher mucin content, and the mucus secretion process can be clearly observed. These results provided a fixation method for paraffin section to facilitate the observation of fish mucous cells and mucus secretion, and a technical support for the subsequent basic research on the regulation of mucin secretion and the mucosal defense role of the mucous cells in fish.