Olfactory Damage and Recovery Histopathology

To determine the olfactory damage and recovery following KDP20 exposure, histopathological analysis, and immunohistochemistry (n = 5 per each group) were performed according to previously reported protocols [50 (link)]. Alcian blue and nuclear fast red (Vector Laboratories. lnc, San Francisco, CA, USA) staining were used to observe the mucin layer, goblet cells, and density. The number of goblet cells per one mm² area, and cell density by number per 100 µm² were quantified in the captured images using Image J 1.52a software. To confirm the expression levels of calretinin, anti-calretinin (Santa Cruz Biotechnology, Dallas, TX, USA) was used. The prepared slides were labeled using an LSAB kit (DAKO, Carpinteria, CA, USA) and observed using a DAB substrate kit (DAKO). The slide images were prepared using a slide scanner (3DHistech, Budapest, Hungary). The optical density of calretinin staining was calculated using Image J 1.52a software.