Vectastain universal and dab reagent kits

In order to ascertain whether MT KO had a lower intrinsic rate of cell proliferation in the olfactory epithelium, PCNA immunohistochemistry was performed on untreated wild-type and MT KO mice. Paraffin embedded sections from untreated wild-type (n=4) and MT KO mice (n=3) were deparaffinized, rehydrated and microwaved in citric acid (10 mM, pH 5.2) for 10 min for antigen retrieval. After washing with 0.01M phosphate buffered saline, sections were blocked with 10% horse serum and then incubated with biotinylated anti-PCNA antibody (NeoMarkers, Fremont, CA; 1:100 dilution) at 4°C overnight in a humidified chamber. Vectastain Universal and DAB reagent kits (Vector, Burlingame, CA) were used to localize antibody binding. PCNA positive cells were counted in the basal layer of the olfactory epithelium of the dorsal meatus of the nasal cavity, and basement membrane lengths were measured using ImageJ software (http://imagej.nih.gov/ij/). Labeling indices were calculated as the number of PCNA positive basal cells divided by the length of the basement membrane.

Zinc gluconate ((ZG) MP Biomedicals LLC) solution was prepared using distilled water and sterilized by syringe-filtering through a 0.2 μm filter disc. Cap’n’Crunch cereal (Quaker Oats Co.), was used for olfactory behavioral assessment. Direct PCR Lysis Reagent (Ear) and Proteinase K solution from Viagen Biotech (Los Angeles, CA) were used for genotyping. Taq PCR Master Mix Kit was purchased from Qiagen (Valencia, CA) for genotyping reactions. Proliferating cell nuclear antigen primary antibody (NeoMarkers, Fremont, CA; 1:100 dilution) and Vectastain Universal and DAB reagent kits (Vector, Burlingame, CA) were used to assess olfactory basal cell proliferation in nasal cavity sections.