Anti calretinin

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.

Calretinin expression in tumor cells was analyzed by flow cytometry (FACSCanto II, BD Biosciences, Heidelberg, Germany) as described previously [8] using the anti‐calretinin (Santa Cruz Biotechnology, Heidelberg, Germany) and anti‐mouse FITC antibody (eBioscience/Thermo Fisher Scientific, Dreieich, Germany). Results were calculated as percentage of calretinin‐positive cells. Apoptosis was measured using the Annexin V FITC apoptosis detection kit (BD Biosciences) according to the manufacturer’s protocol, but using Annexin V APC antibody (BD Biosciences).

Some sections were stained following typical hematoxylin and eosin protocols to observe morphology. Other sections were labeled with immunohistochemistry following typical protocols. Briefly, sections were rehydrated and treated with 3% H₂O₂ in dH₂O to remove endogenous peroxidases. Non-specific binding was blocked with 2% bovine serum albumin and 0.4% Triton X-100 in phosphate-buffered saline (PBS) for 1 h at room temperature. Slides were incubated in a humid chamber at 4 °C for 24 h in anti-calretinin (Santa Cruz Biotechnology, Dallas, TX, USA) at 1:1000 made in blocking solution. Following PBS rinses, sections were incubated at room temperature for 1 h in biotinylated anti-goat IgG (Vector Laboratories, Burlingame, CA, USA) at 1:100 made in blocking solution. Following PBS rinses, sections were treated with ABC solution (Vector Laboratories) for 1.5 h and exposed to diaminobenzidine solution (Vector Laboratories) until sufficient staining was observed. Sections were dehydrated and coverslipped before viewing.