Calci clear rapid

Four mice each of the 4 groups were sacrificed and decapitated 24 hours after the last allergen challenge. The heads were fixed in 4% paraformaldehyde for 3 days at 4℃, washed in running water, decalcified for 3 days with Calci-Clear Rapid (National Diagnostics, Atlanta, GA, USA) at room temperature, dehydrated by passage through a graded alcohol series, and embedded in paraffin block. The blocks were cut into 4-µm-thick sections and stained with hematoxylin and eosin to evaluate the general morphology and number of eosinophils in the lamina propria of the nasal mucosa. The average number of cells was counted in 4 areas around the nasal septa in 50×50-µm areas under a light microscope (×200). The individual who counted eosinophils was blinded to the animals' group assignments.

The Sprague-Dawley rats were anesthetized using a mixture of Zoletil and Rompun solution (9:1 ratio, 1 μl/g, i.m.) and then transcardially perfused with cold saline (0.9% NaCl) and fixed with prechilled 4% paraformaldehyde in phosphate-buffered saline (PBS, pH7.4). The OE and OB were post-fixed overnight at 4°C.
Post-fixed OE tissues were treated with a decalcification solution (Calci-Clear Rapid, National diagnostics, GA, USA) for 4 h. After D.W. washing, OE tissues underwent dehydration with ethanol solutions for 2 h (70%, 85%, 95% and 100%), and then soaked in xylene for 4 h, followed by embedding in paraffin over 4 h.
The OB tissues of rat were sunken in 30% (wt/vol) sucrose overnight for cryo-embedding. Tissues were embedded in optimal cutting temperature (OCT) compound (Leica, Germany), and stored at −80°C until use.
The paraffin-molded samples were sectioned (5 μm) with a rotary microtome (Leica, Germany, RM2235), and cryo-embedded OB tissues were serial sectioned using cryostat at 12 μm (Thermo Scientific, Microm HM550). All sliced tissues were attached to the micro slide glasses (Muto Pure Chemicals, Japan).