a488 conjugated goat anti mouse fab fragment antibodies  (Jackson Immuno)

Journal: The Journal of Neuroscience

Article Title: Dopamine Transporter Localization in Medial Forebrain Bundle Axons Indicates Its Long-Range Transport Primarily by Membrane Diffusion with a Limited Contribution of Vesicular Traffic on Retromer-Positive Compartments

doi: 10.1523/JNEUROSCI.0744-20.2020

Figure Lengend Snippet: Labeling of HA-DAT in MFB axons by stereotactic injection of HA11 antibodies in vivo. A, Stereotactic injections of HA11 antibodies into the MFB area were performed as described in Materials and Methods. B, Schematics of the experimental protocol for labeling plasma membrane and intracellular HA-DAT. Injected antibody binds to a fraction of plasma membrane HA-DAT and internalizes during animal recovery after injections (∼ 2 h). Animals are then killed, brains are dissected, and sagittal slices are prepared and fixed. Fixed slices are incubated with the secondary A488-conjugated donkey anti-mouse-IgG antibody (αM-A488) at 4°C to label HA11:HA-DAT complexes remaining on the surface of neurons. Following permeabilization with Triton X-100 (TX-100), slices are incubated with rabbit monoclonal anti-HA antibody (RαHA) to label intracellular HA-DAT as well as cell-surface HA-DAT that was not occupied by HA11. In the same incubation, goat polyclonal VPS35 antibody (not shown in the protocol scheme) is included. Secondary donkey anti-rabbit and anti-goat antibodies conjugated with Cy3 and Cy5 are finally used to detect RαHA and VPS35 antibodies, respectively. C, 3D stacks of x-y confocal images were acquired from slices in 35 mm MatTek dishes through 488 nm (green, HA11), 561 nm (red, RαHA), and 640 nm (cyan, VPS35) channels. Maximum intensity projections of three consecutive x-y confocal sections are shown. In the merge A488/Cy3-2 image, A488 fluorescence (HA11, surface HA-DAT) was shifted 4 voxels relative to Cy3 fluorescence (RαHA, total HA-DAT), as shown by an arrow for better visualization of highly similar patterns of A488 and Cy3 fluorescence. Colocalization of 488 nm (plasma membrane HA-DAT) and 561 nm (total HA-DAT) channel fluorescence was also estimated by calculating Pearson correlation coefficient (PCC) using SlideBook6 (n = 6). Insets, High-magnification images of the region indicated by white rectangle in the merge image of HA11, RαHA, and VPS35 fluorescence. Small arrows indicate VPS35 puncta in the axon that do not colocalize with HA-DAT puncta. Large arrow indicates an example of HA-DAT (RαHA) localization in VPS35 containing endosome that lacks HA11. Scale bar, 10 µm.

Article Snippet: A488-conjugated goat anti-mouse Fab fragment antibodies (115-547-003) and Cy3-conjugated goat anti-mouse Fcγ-specific Fab fragment antibodies were from Jackson ImmunoResearch Laboratories (115-167-185).

Techniques: Labeling, Injection, In Vivo, Incubation, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Dopamine Transporter Localization in Medial Forebrain Bundle Axons Indicates Its Long-Range Transport Primarily by Membrane Diffusion with a Limited Contribution of Vesicular Traffic on Retromer-Positive Compartments

doi: 10.1523/JNEUROSCI.0744-20.2020

Figure Lengend Snippet: HA-DAT diffusion parameters and internalization in MFB and striatal dopaminergic axons measured in acute sagittal slices. A–D, Acute sagittal brain slices were incubated with the HA11 antibody and Fab fragments of secondary A488-conjugated anti-mouse-IgG antibody at 37°C or room temperature (22°C). Time-lapse images were acquired every 3-5 s at 37°C or 22°C before and after bleaching a small region of an axon. A, Examples of HA-DAT labeling of MFB and striatum in live slices. Scale bars, 10 µm. B, Selected time frames from the time-lapse sequence before and after bleaching of a region of axons in the MFB and striatal areas. Scale bars, 5 µm. C, D, Quantification of the diffusion coefficient (D) and Mf from three independent series of FRAP experiments exemplified in B. Each data point represents calculations of an independent FRAP time-lapse sequence. Differences between D values in MFB and dStr were significant at both 37°C and 22°C (p = 0.012 and p = 0.008, respectively), whereas differences between D values measured at 37°C versus 22°C were not significant. Differences between Mf values were not significant for all variants. One-way ANOVA with Tukey's multiple comparison test was used. E, Internalization assay in acute slices performed under condition recapitulating FRAP measurements at 37°C. Slices were incubated with HA11 antibodies and then Cy3-conjugated Fab fragment of goat anti-mouse Fcγ-specific antibodies at room temperature. After 40 min incubation at 37°C, slices were incubated in nACSF with 5 µg/ml A488-conjugated Fab fragment of goat anti-mouse-IgG antibodies for 30 min at 4°C to label cell-surface HA11:HA-DAT complexes. Slices were then fixed with 4% PFA. F, 3D imaging was performed through 561 nm (red, Cy3; surface plus internalized HA-DAT) and 488 nm (green, A488; surface HA-DAT) channels. Maximum intensity projection images of z stack of 8 consecutive confocal sections are presented. In the Merge2 image, A488 fluorescence was shifted 4 voxels relative to the Cy3 fluorescence as shown by an arrow for better visualization of virtually identical patterns of A488 and Cy3 fluorescence. Insets, High-magnification images of regions indicated by white rectangles. Scale, 10 µm.

Article Snippet: A488-conjugated goat anti-mouse Fab fragment antibodies (115-547-003) and Cy3-conjugated goat anti-mouse Fcγ-specific Fab fragment antibodies were from Jackson ImmunoResearch Laboratories (115-167-185).

Techniques: Diffusion-based Assay, Incubation, Labeling, Sequencing, Imaging, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Dopamine Transporter Localization in Medial Forebrain Bundle Axons Indicates Its Long-Range Transport Primarily by Membrane Diffusion with a Limited Contribution of Vesicular Traffic on Retromer-Positive Compartments

doi: 10.1523/JNEUROSCI.0744-20.2020

Figure Lengend Snippet: Labeling of HA-DAT in MFB axons by stereotactic injection of HA11 antibodies in vivo. A, Stereotactic injections of HA11 antibodies into the MFB area were performed as described in Materials and Methods. B, Schematics of the experimental protocol for labeling plasma membrane and intracellular HA-DAT. Injected antibody binds to a fraction of plasma membrane HA-DAT and internalizes during animal recovery after injections (∼ 2 h). Animals are then killed, brains are dissected, and sagittal slices are prepared and fixed. Fixed slices are incubated with the secondary A488-conjugated donkey anti-mouse-IgG antibody (αM-A488) at 4°C to label HA11:HA-DAT complexes remaining on the surface of neurons. Following permeabilization with Triton X-100 (TX-100), slices are incubated with rabbit monoclonal anti-HA antibody (RαHA) to label intracellular HA-DAT as well as cell-surface HA-DAT that was not occupied by HA11. In the same incubation, goat polyclonal VPS35 antibody (not shown in the protocol scheme) is included. Secondary donkey anti-rabbit and anti-goat antibodies conjugated with Cy3 and Cy5 are finally used to detect RαHA and VPS35 antibodies, respectively. C, 3D stacks of x-y confocal images were acquired from slices in 35 mm MatTek dishes through 488 nm (green, HA11), 561 nm (red, RαHA), and 640 nm (cyan, VPS35) channels. Maximum intensity projections of three consecutive x-y confocal sections are shown. In the merge A488/Cy3-2 image, A488 fluorescence (HA11, surface HA-DAT) was shifted 4 voxels relative to Cy3 fluorescence (RαHA, total HA-DAT), as shown by an arrow for better visualization of highly similar patterns of A488 and Cy3 fluorescence. Colocalization of 488 nm (plasma membrane HA-DAT) and 561 nm (total HA-DAT) channel fluorescence was also estimated by calculating Pearson correlation coefficient (PCC) using SlideBook6 (n = 6). Insets, High-magnification images of the region indicated by white rectangle in the merge image of HA11, RαHA, and VPS35 fluorescence. Small arrows indicate VPS35 puncta in the axon that do not colocalize with HA-DAT puncta. Large arrow indicates an example of HA-DAT (RαHA) localization in VPS35 containing endosome that lacks HA11. Scale bar, 10 µm.

Article Snippet: A488-conjugated goat anti-mouse Fab fragment antibodies (115-547-003) and Cy3-conjugated goat anti-mouse Fcγ-specific Fab fragment antibodies were from Jackson ImmunoResearch Laboratories (115-167-185).

Techniques: Labeling, Injection, In Vivo, Clinical Proteomics, Membrane, Incubation, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Dopamine Transporter Localization in Medial Forebrain Bundle Axons Indicates Its Long-Range Transport Primarily by Membrane Diffusion with a Limited Contribution of Vesicular Traffic on Retromer-Positive Compartments

doi: 10.1523/JNEUROSCI.0744-20.2020

Figure Lengend Snippet: HA-DAT diffusion parameters and internalization in MFB and striatal dopaminergic axons measured in acute sagittal slices. A–D, Acute sagittal brain slices were incubated with the HA11 antibody and Fab fragments of secondary A488-conjugated anti-mouse-IgG antibody at 37°C or room temperature (22°C). Time-lapse images were acquired every 3-5 s at 37°C or 22°C before and after bleaching a small region of an axon. A, Examples of HA-DAT labeling of MFB and striatum in live slices. Scale bars, 10 µm. B, Selected time frames from the time-lapse sequence before and after bleaching of a region of axons in the MFB and striatal areas. Scale bars, 5 µm. C, D, Quantification of the diffusion coefficient (D) and Mf from three independent series of FRAP experiments exemplified in B. Each data point represents calculations of an independent FRAP time-lapse sequence. Differences between D values in MFB and dStr were significant at both 37°C and 22°C (p = 0.012 and p = 0.008, respectively), whereas differences between D values measured at 37°C versus 22°C were not significant. Differences between Mf values were not significant for all variants. One-way ANOVA with Tukey's multiple comparison test was used. E, Internalization assay in acute slices performed under condition recapitulating FRAP measurements at 37°C. Slices were incubated with HA11 antibodies and then Cy3-conjugated Fab fragment of goat anti-mouse Fcγ-specific antibodies at room temperature. After 40 min incubation at 37°C, slices were incubated in nACSF with 5 µg/ml A488-conjugated Fab fragment of goat anti-mouse-IgG antibodies for 30 min at 4°C to label cell-surface HA11:HA-DAT complexes. Slices were then fixed with 4% PFA. F, 3D imaging was performed through 561 nm (red, Cy3; surface plus internalized HA-DAT) and 488 nm (green, A488; surface HA-DAT) channels. Maximum intensity projection images of z stack of 8 consecutive confocal sections are presented. In the Merge2 image, A488 fluorescence was shifted 4 voxels relative to the Cy3 fluorescence as shown by an arrow for better visualization of virtually identical patterns of A488 and Cy3 fluorescence. Insets, High-magnification images of regions indicated by white rectangles. Scale, 10 µm.

Article Snippet: A488-conjugated goat anti-mouse Fab fragment antibodies (115-547-003) and Cy3-conjugated goat anti-mouse Fcγ-specific Fab fragment antibodies were from Jackson ImmunoResearch Laboratories (115-167-185).

Techniques: Diffusion-based Assay, Incubation, Labeling, Sequencing, Comparison, Imaging, Fluorescence