unc13a primary antibody  (abberior instruments)

Journal: The Journal of Cell Biology

Article Title: Homeostatic scaling of active zone scaffolds maintains global synaptic strength

doi: 10.1083/jcb.201807165

Figure Lengend Snippet: BRP, Unc13A, and Cac abundance scale in endo and rab3 mutants, while total levels per NMJ remain constant. (A) Representative type Ib boutons immunostained with anti-BRP, anti-Unc13A, and endogenously tagged Ca 2+ channels (Cac sfGFP-N ) in the indicated genotypes (wild type: cac sfGFP-N ; endo : cac sfGFP-N ; endo 1 /endo Δ4 ; vGlut-OE: cac sfGFP-N ; OK371-Gal4 / UAS-vGlut ; rab3 : cac sfGFP-N ; rab3 rup ). Note that BRP and Unc13A are costained at the same NMJ, while Cac images were acquired from a different NMJ of the same genotype. (B) Quantification of mean fluorescence intensity of individual BRP, Unc13A, and Cac sfGFP-N puncta shows no change in endo and vGlut-OE, with a slight but significant increase in rab3 , consistent with no major difference in the density of material within each punctum. (C) Quantification of BRP, Unc13A, and Cac sfGFP-N puncta sum fluorescence intensity reveals a significant reduction in endo , an increase in rab3 , and no change in vGlut-OE, consistent with the observed changes in puncta area for each genotype ( n ≥ 15; one-way ANOVA; Table S1). (D) The total fluorescence intensity of each individual BRP, Unc13A, and Cac sfGFP-N puncta summed across the entire muscle 4 NMJ terminal is unchanged in endo , rab3, and vGlut-OE compared with wild type. Error bars indicate ± SEM ( n ≥ 8; one-way ANOVA; Table S1). *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; ns, not significant. See Materials and methods for more details on mean, sum, and total intensity measurements.

Article Snippet: For STED imaging, BRP and Unc13A primary antibodies were used and goat anti-guinea pig star635 (1:100; 1–0101002; Abberior) and goat anti-mouse Alexa Fluor 594 (1:500; A11032; Thermo Fisher Scientific) were used for secondary antibodies.

Techniques: Fluorescence

Journal: The Journal of Cell Biology

Article Title: Homeostatic scaling of active zone scaffolds maintains global synaptic strength

doi: 10.1083/jcb.201807165

Figure Lengend Snippet: Elastic modularity of AZ nanostructure revealed by STED imaging. (A) Representative STED images of AZs in wild-type, endo RNAi , and rab3 mutant NMJs labeled with anti-BRP and anti-Unc13A. (B–D) BRP (B) and Unc13A (C) ring diameters are reduced in endo RNAi and enhanced in rab3 mutants, while the BRP:Unc13A ratio remains the same (D). (E) Quantal modules of BRP clusters within single AZs are revealed by STED analysis using an averaging algorithm . The three modules of peak frequency are shown for wild type (4, 5, and 6 modules), endo RNAi (2, 3, and 4), and rab3 (8, 10, and 12). (F–H) Quantification of the mean number of BRP (F) and Unc13A (G) modules per AZ is shown, with a reduction in endo RNAi and enhancement in rab3 , while the ratio is largely unchanged (H). (I and J) Cumulative probability curve (I) and binned histogram (J) of BRP modules per NMJ are shown, with a leftward distribution observed in endo RNAi and rightward shift in rab3 mutants. Error bars indicate ± SEM ( n ≥ 53; one-way ANOVA, K-S test for I; Table S1). *, P < 0.05; ****, P < 0.0001; ns, not significant.

Article Snippet: For STED imaging, BRP and Unc13A primary antibodies were used and goat anti-guinea pig star635 (1:100; 1–0101002; Abberior) and goat anti-mouse Alexa Fluor 594 (1:500; A11032; Thermo Fisher Scientific) were used for secondary antibodies.

Techniques: Imaging, Mutagenesis, Labeling

Journal: The Journal of Cell Biology

Article Title: Homeostatic scaling of active zone scaffolds maintains global synaptic strength

doi: 10.1083/jcb.201807165

Figure Lengend Snippet: Manipulating presynaptic cargo transport to NMJ terminals by arl8 modulates the gain of synaptic strength at endo and rab3 NMJs. (A) Representative images of type Ib NMJ boutons immunostained with antibodies that recognize AZ components (BRP, Unc13A), neuronal membrane (HRP), and SV markers (vGlut, Synaptotagmin [SYT], and Synapsin [SYN]) in wild type, arl8 mutants ( arl8 : w ; arl8 e00336 ), and neuronal overexpression of arl8 (arl8-OE: w;OK6-Gal4,UAS-arl8-GFP/+ ). Note that the number of AZs and the intensity of SV markers at NMJ terminals are reduced by loss of arl8 (C; Table S1), while arl8-OE enhances the intensity of AZ and SV markers (C; Table S1). (B) EPSP traces in the indicated genotypes demonstrate that synaptic strength is reduced by loss of arl8 and increased by arl8-OE. (C) Quantification of the indicated values in the three genotypes normalized to wild-type values ( n ≥ 12; one-way ANOVA; Table S1). (D–F) Representative images (D), EPSP traces (E), and quantification (F) for NMJs of endo RNAi and endo RNAi combined with arl8-OE (endo RNAi +arl8-OE: w;OK6-Gal4,UAS-arl8-GFP/+;UAS-endo-RNAi ). arl8-OE enhances synaptic strength and BRP and vGlut intensity above endo RNAi baseline values (note that values are normalized to endo RNAi ; n ≥ 7; Student’s t test; Table S1). (G–I) Representative images (G), EPSP traces (H), and quantification (I) for NMJs of rab3 RNAi , rab3 , and arl8 double mutants ( rab3 ; arl8 : w;rab3 rup ; arl8 e00336 ), and rab3 RNAi combined with arl8-OE (rab3 RNAi +arl8-OE: w;OK6-Gal4,UAS-arl8-GFP/+;UAS-rab3-RNAi ). Note that loss of arl8 reduces BRP puncta number and intensities of BRP and vGlut below rab3 RNAi baseline values, while arl8-OE enhances these values. Values are normalized to rab3 RNAi ( n ≥ 9; one-way ANOVA; Table S1). Error bars indicate ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Article Snippet: For STED imaging, BRP and Unc13A primary antibodies were used and goat anti-guinea pig star635 (1:100; 1–0101002; Abberior) and goat anti-mouse Alexa Fluor 594 (1:500; A11032; Thermo Fisher Scientific) were used for secondary antibodies.

Techniques: Membrane, Over Expression