Fig 1: Expression and localization of Zn2+ transporter ZnT3 in cultured neurons by immunofluorescence. (a) ZnT3 (synaptic vesicle Zn2+ transporter, pseudocolored green or displayed in grayscale below) and Homer1 (post-synaptic density protein, pseudocolored red) are stained at different timepoints in culture. ZnT3 increases in expression over time in culture, and shows synaptic localization starting at DIV 10. Channel intensities of all images are scaled identically. (b) 2D correlation coefficients calculated on raw immunofluorescence images. Smaller correlation coefficients are more likely to represent less colocalization. ZnT3 maintains a high correlation coefficient compared to Synapsin1/2 across the time course, whereas Synapsin1/2 compared to Homer1 and ZnT3 compared to Homer1 decrease over time. Each DIV/protein comparison is one biological replicate, with each dot representing a separate field of view within the sample.
Fig 2: mRNA expression of Znt3 in isolated taste buds. Znt3 expression was examined using semi-quantitative RT-PCR. TB, NC, and CP indicate isolated taste buds, negative control (H2O; template in place of the cDNA), and taste bud-containing epithelial tissues (circumvallate papillae), respectively. Data are presented as a representative image of three independent experiments. Kcnq1, Ntpdase2, Plcb2, Gnat3, and Aadc were used as taste cell markers of types I, II, and III; type I; type II; type II; and type III, respectively.
Fig 3: Immunohistochemical analysis of ZnT3 expression in the cross-sections of rat circumvallate papillae. A) Representative laser scanning microscopy images of immunohistochemistry with ZnT3 in the circumvallate papillae; the nuclei were counterstained with Hoechst 33258 (blue, 10 mg/mL). B) Immunostaining with an anti-ZnT3 antibody (1st Ab) adsorbed by the immunogen peptide containing the same epitope sequence to generate the antibody. C) The 2nd antibody was used alone as a negative control. Data are presented as a representative image of three independent experiments. Scale bar: 50 mm.
Fig 4: Immunohistochemical localization of ZnT3 and NTPDase2, PLC-b2, or AADC in cross-sections cut through the circumvallate papillae. Representative photomicrographs for double staining of ZnT3 (green) and the type I cell marker NTPDase2 (A; red), type II cell marker PLC-b2 (B; red), or type III cell marker AADC (C; red) are shown. Arrowheads show the colocalization of ZnT3 and taste cell markers in the cell bodies of taste cells. Data are presented as a typical image of three independent experiments. Scale bar: 50 mm. Immunopositive rates of ZnT3 in NTPDase2-, PLC-beta2-, and AADC-positive cells (D). Each value represents the mean ± SD (n=3).
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