Fig 1: Lineage tracing of Axin2+ cells in taste papillae and sweat ducts and nails.(a–j) Adult Axin2-CreERT2/tdT R26REYFP mice were tamoxifen treated at P60 and P61 and EYFP expression analysed in fungiform (a,b,e,f) and circumvallate (c,d,g–j) papillae after 40 h, 6 months or 9 months as indicated. KRT14 marks basal cells (a–d); SOX2 marks basal and Type I TB cells (b,d); PLCß2 marks Type II TB cells (e–h); SNAP-25 marks Type III TB cells (i,j). Yellow arrows indicate EYFP+ basal cells; red arrows indicate EYFP+ differentiated TB cells. Both basal and differentiated TB cells remain labelled over 6–9 months. (k–n) Adult Axin2-CreERT2/tdT R26RmTmG mice were treated with tamoxifen on 2 successive days and mGFP expression analysed in cryosectioned circumvallate papillae at 40 h (k,m) and 4.5 months (l,n) as indicated. KRT14 marks basal cells (k,l); ENTP2 marks differentiated Type I TB cells (m,n). Both basal KRT14+ cells (yellow arrows) and differentiated KRT14- cells, including ENTP2+ Type I cells, (red arrows) were labelled at 40 h and maintained label after 4.5 months. (o–r) Sectioned sweat ducts (o,p) and nail (q,r) from AxinCreERT2/tdT R26RmTmG mice tamoxifen treated at P20-21 and analysed at the stages indicated. Yellow arrows indicate KRT14+ basal cells; red arrows indicate KRT6+ sweat duct suprabasal cells (o,p) or KRT14- differentiating nail plate cells (q,r). Insets in q are magnified images of boxed region; lower image in r is a higher magnification photomicrograph of the arrowed region in the upper image. At least three mice were analysed for each tracing condition; =10 labelled taste papillae or sweat gland ducts and =3 nails were analysed for each condition. Scale bar, 10 µm (a–n), 25 µm (o,p), 50 µm (insets in q), 100 µm (q; magnified region (lower image) in r) or 250 µm (upper image in r).
Fig 2: Dermo1-Cre labeled all three types (I, II, III) of taste bud cells in adult mice.RFP+ signals were co-localized with markers for specific taste cell types (white arrowheads), i.e., NTPDaseII for type I cells, a-Gustducin for type II cells and SNAP25 for type III cells in the lingual (A, B, C) and palatal (D) taste buds. White dotted lines demarcate the epithelium from underlying connective tissue. Scale bar: 20 µm for all images (single plane laser-scanning confocal).
Fig 3: Rat SPG immunohistochemistry. a PACAP-38 immunohistochemistry. PACAP-38 was found in neurons (thin arrows), fibers (arrow heads), and very intense in the SGCs (thick arrows). b nNOS was expressed in the cytoplasm of many of the neurons (thin arrows), negative neurons were also found (asterisk). In addition, immunoreactive pearl-like fibers were observed (arrow heads). c VIP immunoreactivity was disclosed in a granular manner close to the neuronal nuclei, resembling endoplasmatic reticulum staining (arrows). d Serotonin receptor 5-HT1B expression was found most neurons (thin arrows) and fibers (arrow heads), in addition to the vessel walls (thick arrows). e 5-HT1D immunoreactivity was seen in many neurons (arrows) and nerve fibers (arrow heads). f 5-HT1F immunoreactivity was seen in many neurons (arrows) and nerve fibers (arrow heads). g SNAP25 immunoreactivity was found in neurons (thin arrows) in the same granular pattern as described for VIP above. In addition, nerve fibers were immunoreactive (arrow heads). h SV2-A immunoreactivity was exclusively found in the SGCs (arrows)
Fig 4: Immunohistochemistry in combination with tracer label suggests that MNTB to MNTB collaterals form functional synapses.A: The compact circular structure labeled in neuron shown in Fig 1B (box). B: Gephyrin staining localized to the cell membrane (arrows) and surrounding extra-nuclear area (arrowheads). C: The overlap of the two channels shown in A and B reveals gephyrin (cyan) juxtaposed to the biocytin labeled axon (magenta, open arrows). D: The presynaptic label SNAP-25 localized at the circumference of the cell soma (not labeled) and E: Gephyrin (arrows and arrowheads as above). F: The overlay of gephyrin (cyan), SNAP-25 (green) and the corresponding axonal ending (magenta) shows very close proximity of neurite with the presynaptic and postsynaptic density (open arrows). G: A third terminal, which appears to form rudimentary fenestrations. H: Gephyrin staining shows a circular shape that suggests a post synaptic cell body. I: The gephyrin label (cyan) seems to be concentrated around the labeled terminal (magenta) that partly encapsulates the putative postsynaptic soma. Scale bars: 10 µm for all panels.
Fig 5: Specific detection of cleaved SNAP-25 neoepitope by SNAP1 antibody. Differentiated SiMa cells were exposed to the indicated doses of either BoNT/A or BoNT/E and lysed 24 h after intoxication. Lysates were subjected to SDS-PAGE and Western-blotted using either anti-SNAP-25 polyclonal antibody or SNAP1.
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