Journal: STAR Protocols

Article Title: A pipeline for STED super-resolution imaging and Imaris analysis of nanoscale synapse organization in mouse cortical brain slices

doi: 10.1016/j.xpro.2023.102707

Figure Lengend Snippet: Colocalization of GluR1 and lysosomal clusters in L1 apical tufts (A) Representative 60× STED image of an Apical Tuft stained with GFP (white, non-STED channel), GluR1 (green), LAMP1 (magenta) (Scale bar = 1.5 μm). (B and C) Yellow and cyan squares show examples of dendritic spines with GluR1 and LAMP1 clusters that colocalize (Left, example 1, yellow asterisk) and that do not (Right, example 2, cyan asterisk). (B) Scale bar = 250 nm. (C) Scale bar = 100 nm. (D) Selected parameters extracted from the analysis pipeline. (E and F) Frequency distribution of GluR1 and LAMP1 puncta volumes. (G) Frequency distribution and scatter plot of minimum distances between colocalized puncta. Schematic showing minimum distance (d) between two puncta. (H) Frequency distribution and scatter plot of overlap volumes between colocalized puncta. Schematic showing overlap volume between two puncta ( Questions 2 and 3 ). L. layer; Decon, deconvolution; Recon, reconstruction.

Article Snippet: The user will need the find Spots Near to Filament Border and Colocalize Spots Imaris XTensions, available on the Imaris open website.

Techniques: Staining

Journal: STAR Protocols

Article Title: A pipeline for STED super-resolution imaging and Imaris analysis of nanoscale synapse organization in mouse cortical brain slices

doi: 10.1016/j.xpro.2023.102707

Figure Lengend Snippet:

Article Snippet: The user will need the find Spots Near to Filament Border and Colocalize Spots Imaris XTensions, available on the Imaris open website.

Techniques: Recombinant, Saline, Plasmid Preparation, Software, Microscopy

Journal: bioRxiv

Article Title: Liquid droplet germ granules require assembly and localized regulators for mRNA repression

doi: 10.1101/382838

Figure Lengend Snippet: GFP reporter transcripts localize to P-granules when tethered (A) Single molecule fluorescent in situ hybridization (smFISH) in nematode germ cells to visualize RNA expression and localization. (B-M) Gonads were extruded from animals harboring the gfp reporter and (B-E) PGL-1::SNAP (n=30); (F-I) PGL-1::SNAP::λN22 (n=27); (J-M) PGL-1 (K126E K129E)::SNAP::λN22 (n=10). Gonads were fixed and imaged for gfp RNA using (B,F,J) smFISH; (C,G,K) GFP protein fluorescence; (D,H,L) DNA (DAPI) and SNAP. The smFISH, DNA and SNAP images are merged in E,I,M. White arrows mark examples of intranuclear puncta; black arrows mark examples of cytoplasmic puncta. Scale bar, 5 µm, for all images, except for 2.5-fold enlarged images in inset. For germline location, see . (N) Quantification of GFP signal in confocal images for the GFP reporter expressed with PGL-1::SNAP (n=23), PGL-1::SNAP::λN22 (n=24), PGL-1 (K126E K129E)::SNAP::λN22 (n=10). A silenced GFP reporter (n=25) served as a negative control. Mean GFP fluorescent signal and standard deviation reported in Arbitrary Units. (O) smFISH and PGL-1::SNAP signal colocalization in germlines expressing the GFP reporter and either PGL-1::SNAP or PGL-1::SNAP::λN22. Reported as the (Sum of smFISH GFP Intensity in SNAP:smFISH colocalized binned signal)/(Total smFISH GFP binned signal). Box plots represent the first and third quartiles, black line is median, whiskers to min and max values. Co-localization greater in germlines expressing PGL-1::SNAP::λN22 ( p <0.01). See Methods for more details.

Article Snippet: The Surface-Surface Colocalization XTension was downloaded and installed from the Bitplane XTension File Exchange at http://open.bitplane.com/tabid/235/Default.aspx?id=111 .

Techniques: In Situ Hybridization, RNA Expression, Fluorescence, Negative Control, Standard Deviation, Expressing

Journal: bioRxiv

Article Title: Liquid droplet germ granules require assembly and localized regulators for mRNA repression

doi: 10.1101/382838

Figure Lengend Snippet: PGL-1-tethered GFP reporter transcripts localize to granules in the presence or absence of WAGO-1 Gonads were extruded from gfp reporter, PGL-1::SNAP::λN22 animals harboring (A-D) wago-1 wild-type (n=22); (E-H) wago-1 null (n=21). Gonads were fixed and imaged for (A,E) gfp RNA using smFISH, (B,F) GFP protein fluorescence, (C,G) DNA (DAPI) and SNAP. gfp RNA smFISH, DNA and SNAP merged in D, H. White arrows mark examples of intranuclear puncta; black arrows mark examples of cytoplasmic puncta. Scale bar, 5 µm, for all images, except for 2.5-fold enlarged images in inset. For germline location, see . (I) smFISH and PGL-1::SNAP signal colocalization in germlines expressing the GFP reporter, PGL-1::SNAP::λN22 in the presence or absence of WAGO-1. Reported as the (Sum of smFISH GFP Intensity in SNAP:smFISH colocalized binned signal)/(Total smFISH GFP binned signal). Box plots represent the first and third quartiles, black line is median, whiskers to min and max values. Co-localization similar in germlines expressing PGL-1::SNAP::λN22 with or without WAGO-1 (p=0.039). See Methods for more details.

Article Snippet: The Surface-Surface Colocalization XTension was downloaded and installed from the Bitplane XTension File Exchange at http://open.bitplane.com/tabid/235/Default.aspx?id=111 .

Techniques: Fluorescence, Expressing

Journal: The EMBO Journal

Article Title: Microglia‐synapse engulfment via PtdSer‐TREM2 ameliorates neuronal hyperactivity in Alzheimer's disease models

doi: 10.15252/embj.2022113246

Figure Lengend Snippet: A Area under curve (AUC) for pHrodo fluorescence signals in primary microglia at t = 3 h, normalized to respective control, post‐application of synaptosomes (SN) from either AD patients or NDC brains, with and without Annexin‐V (AnnxV) pretreatment. Data are normalized to NDC SN. ∼40 microglia per ROI, two ROIs per well, 2–3 wells per experiment, n = 6 human cases for NDC and AD, n = 3 independent experiments. B Representative image of primary microglia simultaneously treated with Aβ oligomer‐bound SN (oAβ‐SN; in pHrodo red [magenta]) and control SN (Ctrl‐SN; in pHrodo deep red [cyan]). Scale bar, 50 μm. C pHrodo fluorescence (a.u.) over 10 h (3–5‐min intervals, SNs added to microglia at t = 0) showing a faster rate of increase of oAβ‐SN compared with Ctrl‐SN. Data are normalized to Ctrl‐SN. ∼40 microglia per ROI, two ROIs per well, 2–3 wells per experiment, n = 4 independent experiments. D Percentage of pHrodo fluorescence of either oAβ‐SN or Ctrl‐SN in microglia at t = 3 h. ∼40 microglia per ROI, two ROIs per well, 2–3 wells per experiment, n = 3 independent experiments. E pHrodo fluorescence with time shown as AUC at 3 h normalized to respective control. AUC of engulfed mouse oAβ‐SN versus Ctrl‐SN with and without AnnxV pretreatment. F Time‐lapse images of primary Homer1‐eGFP neurons (green) treated with 50 nM oAβ versus vehicle control. Yellow arrows indicate increasing PSVue550 (magenta) signal on dendritic spines with Aβ oligomer treatment over 45 min. Scale bar, 2 μm. G Relative fold change of colocalized PSVue and Homer1‐eGFP signal over time. Two–three ROIs per experiment, n = 3 independent experiments. H Orthogonal view of AiryScan image showing colocalization of Homer1‐eGFP (green) and PSVue (magenta) at 1‐h post‐treatment with Aβ oligomer. Scale bar, 2 μm. I Quantification of colocalized Homer1‐eGFP with PSVue among total Homer1. Three ROIs per neuron, 4–5 neurons, n = 3 independent experiments. J Quantification of colocalized PSVue with Homer1‐eGFP among total PSVue. Three ROIs per neuron, 4–5 neurons, n = 3 independent experiments. Data information: Data shown as mean ± SEM (A–E) or as mean ± SD (F–J). Each shaded point represents one ROI, and each open point represents the mean of each independent experiment (A, D, E, I, J). Filled point (G) represents the average of all ROIs. One‐way (A and E) or two‐way (G) ANOVA followed by Bonferroni post hoc test, paired (D) or unpaired (I and J) t ‐test. P ‐values shown as * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Source data are available online for this figure.

Article Snippet: Pre‐ and postsynaptic spots within 0.25 μm of a PSVue surface were determined using a MATLAB colocalization script (Spots Close To Surface XTension).

Techniques: Fluorescence

Journal: The EMBO Journal

Article Title: Microglia‐synapse engulfment via PtdSer‐TREM2 ameliorates neuronal hyperactivity in Alzheimer's disease models

doi: 10.15252/embj.2022113246

Figure Lengend Snippet: A Representative image of Homer1‐eGFP neuron used in our studies here (green, left panel). In contrast to an apoptotic neuron (magenta, middle panel), where PSVue signals are observed in whole parts of the dying cell, the neurons used in our studies are nonapoptotic with negligent PSVue signal in the soma (right panel). Scale bar 10 μm. B Super‐resolution Airyscan images of neuronal PSvue (magenta) staining after 1‐h treatment of 50 nM Aβ oligomer. Note PSVue signals in insets 1 and 2 are not random but are colocalized with or in close vicinity to Homer1‐eGFP signal as indicated by yellow arrowheads. Scale bar 10, 2, and 5 μm. C Super‐resolution Airyscan images of PSD95 (cyan), PSVue (magenta) and 6E10 anti‐Aβ antibody (yellow) after 1‐h treatment with Aβ oligomer. Yellow arrowheads indicate the spines showing colocalized signal of PSD95, PSVue, and 6E10. Magnified orthogonal view of a single spine with PSD95, PSVue, and 6E10 colocalization in the inset. Scale bar 2 and 0.5 μm.

Article Snippet: Pre‐ and postsynaptic spots within 0.25 μm of a PSVue surface were determined using a MATLAB colocalization script (Spots Close To Surface XTension).

Techniques: Staining

Journal: The EMBO Journal

Article Title: Microglia‐synapse engulfment via PtdSer‐TREM2 ameliorates neuronal hyperactivity in Alzheimer's disease models

doi: 10.15252/embj.2022113246

Figure Lengend Snippet: A Super‐resolution images from the hippocampal CA1 stratum radiatum of 6‐month‐old NL‐F KI and NL‐F KI; Trem2 R47H KI mice immunostained for presynaptic Synaptotagmin 1/2 (Syt1/2, red) and postsynaptic Homer1 (green). PSVue 643 (magenta) is ICV injected. Upper panels show Syt1/2, Homer1, and PSVue. Lower panels show Homer1 and PSVue only. Scale bar 1 μm. B PSVue volume represented in μm 3 . Three ROIs per animal, n = 3–4 per genotype. C Percentage of synaptic Synaptotagmin 1/2 puncta within 0.25 μm of PSVue showing increased percentage of PSVue + synapses in NL‐F KI; Trem2 R47H KI mice compared with NL‐F KI. Three ROIs per animal, n = 3–4 animals per genotype. D Percentage of PSVue volume within 0.25 μm of synaptic Synaptotagmin 1/2 puncta. Three ROIs per animal, n = 3–4 animals per genotype. E Super‐resolution images from the hippocampal CA1 dentate gyrus hilus of 4‐month‐old WT and J20 Tg mice immunostained for pre‐Synaptotagmin 1/2 (Syt1/2, red) and postsynaptic Homer1 (green). PSVue 643 (magenta) is ICV injected. Top panels show Syt1/2, Homer1, and PSVue. Bottom panels show Homer1 and PSVue only. Insets show either triple (Syt1/2, Homer1 and PSVue; top panel) or double colocalization (Homer1 and PSVue; bottom panel). Scale bar 1 μm. F PSVue total volume per ROI (7,500 μm 3 ) showing increased PSVue in J20 Tg mice compared with WT. Three ROIs per animal, n = 3–4 animals per genotype. G Percentage of synaptic Homer1 puncta within 0.25 μm of PSVue showing an increase in PSVue + synapses in J20 Tg compared with WT. Three ROIs per animal, n = 3–4 animals per genotype. H Percentage of synaptic Synaptotagmin 1/2 puncta within 0.25 μm of PSVue showing an increase in PSVue + synapses in J20 Tg compared with WT. Three ROIs per animal, n = 3–4 animals per genotype. I Percentage of PSVue volume within 0.25 μm of synaptic Homer1 puncta. Three ROIs per animal, n = 3–4 animals per genotype. Data information: Data shown as mean ± SEM. Each shaded point represents one ROI, and each open point represents the average per experimental replicate. Two‐way ANOVA followed by Bonferroni's post hoc test. P ‐values shown as ns P > 0.05; * P < 0.05; ** P < 0.01.

Article Snippet: Pre‐ and postsynaptic spots within 0.25 μm of a PSVue surface were determined using a MATLAB colocalization script (Spots Close To Surface XTension).

Techniques: Injection