Extended Data Fig. 1: The modified FingR(PSD95)-GFP construct labels synapses in vivo.
From: Sleep pressure modulates single-neuron synapse number in zebrafish
a-a”, Maximum projection (Z-stack, ~10 μm) of anti-MAGUK immunohistochemistry and endogenous fluorescence of FingR(PSD95)-GFP in the spinal cord of 2 dpf Tg(mnx1:Gal4) larvae. Examples of FingR(PSD95)+ puncta co-labelled by anti-MAGUK are indicated by white arrowheads; an example of a FingR(PSD95)+ not labelled by anti-MAGUK is indicated by the blue arrowhead. b-b”’, Higher magnification (white box from a) depicting how sectional grey values for each synapse were obtained. b, The FingR(PSD95)-GFP channel showing part of a neuron with its nucleus (asterisk) and synaptic puncta (green). Dotted lines indicate example cross-sectional areas obtained for each synapse. b’, Anti-MAGUK puncta of the same neuron. b”,b”’, FingR(PSD95)-GFP and MAGUK channels merged, with examples of cross-sections 1–4. c, Examples of normalized cross-sectional grey values for anti-MAGUK signals and FingR(PSD95)-GFP signal for the same puncta (numbered 1–4 in b”’). Three examples in which FingR(PSD-95)-GFP co-localized with anti-MAGUK signals (#1–3) and one example (#4) where a FingR(PSD-95)-GFP punctum did not co-localize with MAGUK. See Methods for details. d, Percentage of FingR(PSD-95)-GFP synapses that co-localized with anti-MAGUK+ puncta (blue). As a control for chance co-localization, the calculation was repeated on images in which the anti-MAGUK image was rotated by 90° relative to the FingR(PSD-95)-GFP channel. ****P = 1.1 × 10−83 Chi-square. e, Histogram of the distance between all co-localized FingR(PSD95)-GFP and anti-MAGUK cross-sectional grey value peaks. f-g, The intensity and Full Width Half Max (FWHM) of FingR(PSD95)-GFP and anti-MAGUK puncta are weakly, but significantly, positively correlated. Blue and red lines depict the linear regression curve and 95% CI for the colocalized and non-colocalized populations, respectively. n = 540 puncta, 5 fish (data as in d). h, Percentage of anti-MAGUK+ puncta that co-localized with FingR(PSD-95)-GFP synapses (blue). As a control for chance co-localization, the calculation was repeated on images in which the FingR(PSD-95)-GFP image was rotated by 90° relative to the anti-MAGUK channel. ****P = 3.1 × 10−14 Chi-square. i, Histogram of the distance between co-localized anti-MAGUK and FingR(PSD95)-GFP cross-sectional grey value peaks. Scale bar: 5 μm (a-b”’).
