Journal: STAR Protocols

Article Title: Protocol for SUM-PAINT spatial proteomic imaging generating neuronal architecture maps in rat hippocampal neurons

doi: 10.1016/j.xpro.2025.103637

Figure Lengend Snippet: Workflow for SUM-PAINT sample preparation and imaging Steps describing labeling of the sample and SUM-PAINT labeling and imaging workflow. After primary and secondary antibody and nanobodies are preincubated in sets of 6 targets the pooled antibodies are incubated onto the sample. With secondary hybridization of barcoding round 1 the imaging can be conducted in sequential fashion using speed-optimized R-sequences. When the imaging is done signal extinction is done via blocking of the docking strands by the hybridization of a full complement. Once this step is completed the next barcoding round can start until all targets are acquired and the neuronal atlas can be reconstructed.

Article Snippet: Prepare premixed incubation stocks for primary antibodies and secondary nanobodies for the first barcoding round (6 targets). a. Pipette 10 μL of antibody incubation buffer in a 500 μL Eppendorf tube, for each target respectively. b.

Techniques: Sample Prep, Imaging, Labeling, Incubation, Hybridization, Blocking Assay

Journal: STAR Protocols

Article Title: Protocol for SUM-PAINT spatial proteomic imaging generating neuronal architecture maps in rat hippocampal neurons

doi: 10.1016/j.xpro.2025.103637

Figure Lengend Snippet: Problems and their possible solutions (A) Three TIRF angles for image acquisition of synaptic targets are shown in ‘Picasso: Localize’ with the left image displaying a too low HILO sheet for image acquisition indicated uneven illumination and a clear loss of localization on the right side of the image. The middle image shows the perfect HILO illumination showing evenly distributed localizations throughout the field of view and continuous sampling. The right image shows a too high HILO with substantially higher noise and highly reduced sampling. HILO adjustment in detail in . (B) Three imager concentrations (20 pM, 60 pM, 200 pM, , , and ) for Golga5 acquisition are shown. 20 pM as imager concentration is too low indicated by very few blinking events. 60 pM is the optimal imager concentration for imaging Golga5 as the sample is regularly sampled with rarely overlapping point spread functions (PFS). 200 pM are substantially too high as several PSFs overlap. This imaging is useful for determining the perfect plane for target acquisition but should be replaced with a lower concentration for higher resolution. (C) Nanobody crosstalk qualitative comparison after 1 and 3 h and overnight (approximately 16 h) incubation of a sample with preincubated BC6 2ry nanobody to a 1ry antibody and BC22 nanobody in solution. Imaging of the same region indicates that there is no visible crosstalk after 3 h but a minor part appears with overnight (approximately 16 h) incubation. (D) Comparison of emission detection before and after extinction. The top row shows SUM-PAINT imaging before extinction. The raw image as well as the PSF detection show a highly sampled structure, which is reconstructed afterward. In the bottom row the same region after extinction by blocking shows only gold detection and the reconstructed image shows no trace of an underlying structure. (E) Image alignment workflow in ‘Picasso: Render’. The raw overlay of two regions shows an offset of a few 100 nm. Image alignment by Picasso results in nanometer precise overlay.

Article Snippet: Prepare premixed incubation stocks for primary antibodies and secondary nanobodies for the first barcoding round (6 targets). a. Pipette 10 μL of antibody incubation buffer in a 500 μL Eppendorf tube, for each target respectively. b.

Techniques: Sampling, Concentration Assay, Imaging, Comparison, Incubation, Blocking Assay

Journal: STAR Protocols

Article Title: Protocol for SUM-PAINT spatial proteomic imaging generating neuronal architecture maps in rat hippocampal neurons

doi: 10.1016/j.xpro.2025.103637

Figure Lengend Snippet: Antibody incubation buffer

Article Snippet: Prepare premixed incubation stocks for primary antibodies and secondary nanobodies for the first barcoding round (6 targets). a. Pipette 10 μL of antibody incubation buffer in a 500 μL Eppendorf tube, for each target respectively. b.

Techniques: Incubation, Concentration Assay