Figure S5. Zn 2 influx promotes... | Rockefeller University Press

Figure S5.

Zn2+influx promotes detachment of endogenous neuronal Tau but no changes in microtubule acetylation in situ. (A) Representative immunofluorescence micrographs (15 × 15 stitched image grid, 5% overlap) of methanol-fixed primary rat hippocampal neurons depolarized with 50 mM KCl in the absence (top) or presence (bottom) of 100 µM ZnCl2. Fixed cells were incubated with anti-β-tubulin monoclonal antibodies (magenta, left), anti-Tau polyclonal antibodies (green, left center), DAPI (cyan, right center), and channels are shown merged (right). Scale bar = 200 µm. (B and C) Representative immunofluorescence micrographs at (B) low magnification or (C) high magnification, of methanol-fixed primary rat hippocampal neurons depolarized with 50 mM KCl in the absence (top) or presence (bottom) of 100 µM ZnCl2. Fixed cells were incubated with anti-β-tubulin monoclonal antibodies (magenta, left), anti-acetylated α-tubulin polyclonal antibodies (green, left center), DAPI (cyan, right center), and channels are shown merged (right). Scale bar = 10 µm. (D) Mean fluorescence intensity of AlexaFluor 488 secondary antibody-labeled acetylated α-tubulin (normalized to AlexaFluor 555-conjugated β-tubulin monoclonal antibodies) in neurons depolarized with 50 mM KCl in the absence or presence of 100 µM ZnCl2 (50 mM KCl: n = 180 linear segments from three biological replicates; 50 mM KCl and 100 µM ZnCl2: n = 180 linear segments from three biological replicates). All experiments were performed in the absence of extracellular Ca2+.

Zn ²⁺ influx promotes detachment of endogenous neuronal Tau but no changes in microtubule acetylation in situ. (A) Representative immunofluorescence micrographs (15 × 15 stitched image grid, 5% overlap) of methanol-fixed primary rat hippocampal neurons depolarized with 50 mM KCl in the absence (top) or presence (bottom) of 100 µM ZnCl₂. Fixed cells were incubated with anti-β-tubulin monoclonal antibodies (magenta, left), anti-Tau polyclonal antibodies (green, left center), DAPI (cyan, right center), and channels are shown merged (right). Scale bar = 200 µm. (B and C) Representative immunofluorescence micrographs at (B) low magnification or (C) high magnification, of methanol-fixed primary rat hippocampal neurons depolarized with 50 mM KCl in the absence (top) or presence (bottom) of 100 µM ZnCl₂. Fixed cells were incubated with anti-β-tubulin monoclonal antibodies (magenta, left), anti-acetylated α-tubulin polyclonal antibodies (green, left center), DAPI (cyan, right center), and channels are shown merged (right). Scale bar = 10 µm. (D) Mean fluorescence intensity of AlexaFluor 488 secondary antibody-labeled acetylated α-tubulin (normalized to AlexaFluor 555-conjugated β-tubulin monoclonal antibodies) in neurons depolarized with 50 mM KCl in the absence or presence of 100 µM ZnCl₂ (50 mM KCl: n = 180 linear segments from three biological replicates; 50 mM KCl and 100 µM ZnCl₂: n = 180 linear segments from three biological replicates). All experiments were performed in the absence of extracellular Ca²⁺.