Journal: bioRxiv

Article Title: Lead as a toxic environmental toxicant in models of synucleinopathies

doi: 10.1101/2024.10.10.617554

Figure Lengend Snippet: A. Representative images of the GFAP (1:1000)- stained astrocytes and aSynuclein (1:200). B. Histogram of the percentage of GFAP-positive cells’ quantification. C. Histogram of GFAP fluorescence intensity quantification. D. Histogram of GFAP cell area quantification. E. Histogram of aSyn fluorescence intensity quantification. F. Histogram of paSyn S129 fluorescence intensity quantification. Images were acquired on a confocal point scanning microscope, (Zeiss LSM 900 with Airyscan 2), with a 20x objective for whole dentate gyrus imaging and a 63x objective for zooming in on the astrocytic cells. The scale bar is 100 µm or 50 µm for stained images. Values are the mean ± SD for n=3-8 for all groups. The symbols denote statistically significant differences inter groups: Ctrl PBS vs IntPb PBS/PerPb PBS; Ctrl aSyn vs IntPb aSyn/PerPb aSyn - *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; Ctrl PBS vs IntPb aSyn/PerPb aSyn; Ctrl aSyn vs IntPb PBS/PerPb PBS - # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001; Ctrl PBS vs Ctrl aSyn/ IntPb PBS vs IntPb aSyn/ PerPb PBS vs PerPb aSyn - $ p < 0.05, $$ p < 0.01, $$$ p < 0.001, $$$$ p < 0.0001; one-way ANOVA, Tuckey’s multiple comparison test.

Article Snippet: Succinctly, free-floating sections were washed with TBS 1x (PanReac/AppliChem, Germany), permeabilized with 3% Triton X 100 solution for 15min (Carl Roth, Germany) and blocked with 5% Goat Serum (BioWest, France) and 1% Bovine serum (VWR, USA) for 1 h. The freely floating tissue were then incubated with specific combinations of primary antibodies [astrocytes - glial fibrillary acidic protein - GFAP (chicken, ab4674, abcam, 1:500), alpha synuclein (mouse, 610787, BD Biosciences), phosphorylated alpha synuclein S129 – paSyn S129 (rabbit, SMC-600, StressMarq), Tyrosine Hydroxylase - TH (mouse, MAB5280, Milipore) and Tyrosine Hydroxylase – TH (rabbit, AB152, Milipore)] overnight at 4°C on a shaker.

Techniques: Staining, Fluorescence, Microscopy, Imaging, Comparison

Journal: bioRxiv

Article Title: Lead as a toxic environmental toxicant in models of synucleinopathies

doi: 10.1101/2024.10.10.617554

Figure Lengend Snippet: A. Representative images of the TH (1:500)-stained dopaminergic neurons and aSyn (1:200) in the substantia nigra. B. Representative images of the TH (1:500)-stained dopaminergic neurons and paSyn S129 (1:200) in the substantia nigra. C. Histogram of the quantification of TH+ve cells. D. Histogram of quantification of aSyn fluorescence intensity in substantia nigra. E. Histogram of quantification of aSyn fluorescence intensity in striatum. F. Histogram of quantification of paSyn S129 fluorescence intensity in substantia nigra G. Histogram of quantification of paSyn S129 fluorescence intensity in striatum. Images were acquired on a confocal point scanning microscope, (Zeiss LSM 900 with Airyscan 2), with a 10x objective. The scale bar is 100 µm for stained images. Values are the mean ± SD for n=4-8 for all groups. The symbols denote statistically significant differences inter groups: Ctrl PBS vs IntPb PBS/PerPb PBS; Ctrl aSyn vs IntPb aSyn/PerPb aSyn - *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; Ctrl PBS vs IntPb aSyn/PerPb aSyn; Ctrl aSyn vs IntPb PBS/PerPb PBS - # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001; one-way ANOVA, Tuckey’s multiple comparison test.

Article Snippet: Succinctly, free-floating sections were washed with TBS 1x (PanReac/AppliChem, Germany), permeabilized with 3% Triton X 100 solution for 15min (Carl Roth, Germany) and blocked with 5% Goat Serum (BioWest, France) and 1% Bovine serum (VWR, USA) for 1 h. The freely floating tissue were then incubated with specific combinations of primary antibodies [astrocytes - glial fibrillary acidic protein - GFAP (chicken, ab4674, abcam, 1:500), alpha synuclein (mouse, 610787, BD Biosciences), phosphorylated alpha synuclein S129 – paSyn S129 (rabbit, SMC-600, StressMarq), Tyrosine Hydroxylase - TH (mouse, MAB5280, Milipore) and Tyrosine Hydroxylase – TH (rabbit, AB152, Milipore)] overnight at 4°C on a shaker.

Techniques: Staining, Fluorescence, Microscopy, Comparison