Fig 2: X10ht-STED imaging of isolated synaptic vesicles. (A) Representative confocal and STED images of one possible combination of amplification systems for SYP1, SYT1 and VGLUT1, revealed by AS580, AF488 and AS635P, respectively. (C) The protein targets and their amplification systems are combined with another selection of fluorophores. (B, D) The fluorescence intensities of all channels were analyzed, and indicated a sufficient signal-to-noise ratio for vesicle identification. N = 74 ROIs from 27 images in B, and N = 33 ROIs from 20 images in (D). (E) Line scans were drawn over the spots indicated in zoom panels in (C). The respective full widths at half maximum (FWHM) are indicated. (F) An analysis of spot sizes of different fluorophores with N = 72 analyzed AF488 spots from 20 images, N = 78 AS635P spots from 18 images, and N = 34 Cy3 spots from 14 images from two different experiments. Data for B, D and F are presented as individual data points, mean ± SD.

Fig 3: Applicability of nanobodies in X10ht, compared to homogenization with proteinase K (original X10 protocol). (A) Exemplary epifluorescence images of hippocampal cultured neurons immunostained with antibodies (Ab) for the postsynaptic protein SHANK2 and the presynaptic protein synaptotagmin1 (SYT1), revealed by secondary antibodies (AbSHANK2 + AbAF488) or secondary nanobodies (Nb) (AbSYT1 + NbAF546). (B) A pronounced increase in fluorescence intensity in both immunostainings is obtained when samples are autoclaved (X10ht) compared with the original X10 protocol. N = 6 images with several ROI analyzed for AbSHANK2 + AF488 X10 and AbSYT1 + NbAF546 X10, and N = 5 images with several ROI analyzed for AbSHANK2 + AF488 X10ht and AbSYT1 + NbAF546 X10ht. (C) Exemplary images of SYT1 antibody immunostainings, revealed by a secondary antibody (AbSYT1 + Ab AF546), in parallel with labeling for the vesicular glutamate transporter 1 (VGLUT1) with primary nanobodies coupled to AF488 (NbVGLUT1-AF488). (D) The quantification of the fluorescence signal revealed significantly increased intensities with X10ht. N = 6 images with several ROI analyzed for AbSYT1 + AbAF546 X10 and NbVGLUT1-AF488 X10, and 8 images with several ROI analyzed for AbSYT1 + AbAF546 X10ht and NbVGLUT1-AF488 X10ht from 2 gels. Data are presented as single data points, mean ± SD.