Journal: Protein Science : A Publication of the Protein Society

Article Title: Rapid directed molecular evolution of fluorescent proteins in mammalian cells

doi: 10.1002/pro.4261

Figure Lengend Snippet: Intracellular brightness and photostability of mCardinal, TagRFP658, and emiRFP2 in live cultured hippocampal mouse neurons and in vivo in zebrafish larvae. (a,b) Representative fluorescence images of primary cultured mouse hippocampal neurons expressing TagRFP658 at (a) 14 and (b) 23 days in vitro (DIV; n = 53 and 33 neurons, respectively, from two independent cultures). Imaging condition: excitation 631/28 nm from an LED, emission 664LP. (c) Representative light sheet image of head of zebrafish larvae at 4 days postfertilization expressing TagRFP658 in neurons ( n = 10 fish from two independent injections). Imaging conditions: excitation 638 nm from a laser, emission 665LP. (i, ii) High‐magnification images of the respective regions shown in white boxes in e. Scale bars, 50 μm. (d) Relative fluorescence of cultured mouse hippocampal neurons expressing mCardinal and TagRFP658 ( n = 78 and 85 neurons, respectively, from two independent cultures for each protein; one‐way analysis of variance [ANOVA]). Imaging conditions as in (a). Box plots with notches are used in this figure (see Figure for the full description). (e) Raw photobleaching curves for mCardinal (dashed line) and TagRFP658 (solid line) in primary cultured mouse hippocampal neurons ( n = 9 and 7 neurons, respectively, from one culture each; one‐way ANOVA). Imaging condition: excitation 631/28 nm from an LED at 70 mW/mm 2 , emission 664LP. (f) Representative fluorescence images of cells transfected with pAAV‐CAG‐mCardinal‐P2A‐GFP (top), pAAV‐TagRFP658‐P2A‐GFP (middle), and pAAV‐emiRFP2‐P2A‐GFP ( n = 39, 33, and 41 neurons from three, two, and three independent transfections from one culture each for mCardinal, TagRFP658, and emiRFP2, respectively, for Cy5 channel and n = 15 and neurons from one independent transfection from one culture each for mCardinal and emiRFP2, respectively, for Cy5.5 channel). Imaging conditions: Cy5 channel: excitation 635/22 nm from 637 nm laser, emission 730/140 nm; Cy5.5 channel: excitation 680/13 nm from 680 nm laser, emission 710 LP; GFP channel: excitation 478/24 nm for an LED; emission 535/46 nm. Images in Cy5 and Cy5.5 were taken under matching excitation intensity (66 mW/mm 2 ) and the same exposure time (100 ms). The dynamic range of fluorescence intensity in Cy5 and Cy5.5 channels are identical across all images. Scale bar, 20 μm. (g) Near‐infrared (NIR)‐to‐green fluorescence ratio for mCardinal, TagRFP658, and emiRFP2 for the experiment shown in (f). (h) Intracellular photostability of mCardinal, TagRFP658, and emiRFP2 in Cy5 and Cy5.5 channels ( n = 8, 7, and 9 neurons from three, two, and three independent transfections from one culture each for mCardinal, TagRFP658, and emiRFP2, respectively, under Cy5 excitation and n = 5 neurons from one transfection from one culture for emiRFP2 under Cy5.5 excitation). Imaging conditions the same as in (a)

Article Snippet: Mouse primary hippocampal neurons were prepared from postnatal day 0 or 1 Swiss Webster (Taconic) mice (both male and female mice were used) and cultured as previously described.

Techniques: Cell Culture, In Vivo, Fluorescence, Expressing, In Vitro, Imaging, Transfection

Journal: Cell

Article Title: Multimodal charting of molecular and functional cell states via in situ electro-sequencing

doi: 10.1016/j.cell.2023.03.023

Figure Lengend Snippet: (A) Schematics illustrating in situ electro-seq of neural patches. (B) Representative voltage traces showing spike-bursting dynamics of mouse hippocampal neurons (i) with the bursting activity (ii) and single spike train (iii) highlighted. (C) Detected spike trains from continuous recording (left panel) and single spikes (right panel) from the dashed box highlighted region. (D) Overlapped 3D cell-type and electrode maps. Grey color labels each individual electrode. (E) Identified electrically recorded neurons. Colors label spikes identified from each neuron highlighted by white arrows. Zoomed-in image shows one neuron that was simultaneously recorded by four electrodes. (F) UMAP visualizations of all the sequenced cells. (G) Heatmap showing the electrophysiological features and marker gene expression profiles. (H) Box and dot plots showing the peak-trough ratio between excitatory and inhibitory neurons. n = 20 for excitatory neurons, n = 15 for inhibitory neurons, ** p < 0.01, two-tailed, unpaired t test.

Article Snippet: Mouse primary hippocampal neurons , Broad Institute of MIT and Harvard , C57BL/6 mice.

Techniques: In Situ, Activity Assay, Marker, Gene Expression, Two Tailed Test

Journal: Cell

Article Title: Multimodal charting of molecular and functional cell states via in situ electro-sequencing

doi: 10.1016/j.cell.2023.03.023

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse primary hippocampal neurons , Broad Institute of MIT and Harvard , C57BL/6 mice.

Techniques: Recombinant, Microscopy, In Situ, Sequencing, Software

Journal: iScience

Article Title: Phosphorylation of presynaptic PLPPR3 controls synaptic vesicle release

doi: 10.1016/j.isci.2025.113435

Figure Lengend Snippet: PLPPR3 and BASP1 localize to the presynaptic terminal (A) Phosphorylation of PLPPR3 S351 can be triggered in neurons. DIV8 primary hippocampal neurons were stimulated with Forskolin (30 μM, 5 min), and analyzed by western blot using indicated antibodies. (B) Quantification of the results in A ( n = 3). Data are represented as mean ± SEM. ∗∗∗∗ p < 0.0001, unpaired t test. (C) Localization of PLPPR3 and pS351 to synaptosomal fractions. Crude synaptosomes were prepared from adult mouse brain and analyzed by western blot. S2, cytosolic fraction; P2, crude synaptosomes; SYP1, Synaptophysin1. (D) Localization of PLPPR3 to presynaptic terminals in DIV16 WT primary hippocampal neurons analyzed by STED microscopy. PLPPR3 was stained with our custom made anti-PLPPR3 antibody in combination with anti-VGLUT1. (E) Co-localization of Synaptophysin1 and PLPPR3 ICDm following overexpression in primary hippocampal neurons. Neurons were transfected with recombinant proteins at DIV1 and analyzed at DIV7. (F) Quantification of PLPPR3 ICDm clusters inside Synaptophysin1-positive synapses. Synaptophysin1 was co-expressed with PLPPR3 ICDm or PLPPR3 ICDm S351A. Graph shows PLPPR3 ICDm (WT or S351A) in Synaptophysin1-positive synapses. N = 3, n ≥ 7 neurons. Data are represented as mean ± SEM. (G) Localization of BASP1 to presynaptic terminals in DIV16 WT primary hippocampal neurons analyzed by STED microscopy. BASP1 was stained with the custom made anti-BASP1 antibody in combination with anti-VGLUT1. (H) Co-localization of Synaptophysin1 and BASP1 in WT hippocampal neurons. (I) Co-localization of PLPPR3 ICDm and BASP1 in Plppr3−/− hippocampal neurons. In (H) and (I), neurons were transfected with recombinant proteins at DIV1 and imaged at DIV7. Scale bars, 5 μm. (J) Distance of BASP1 clusters to nearest VGLUT1 clusters (pre-synapses) in WT and PLPPR3 −/− neurons, and in PLPPR3 −/− neurons expression PLPPR3 ICDm or PLPPR3 ICDm S351A. Data are represented as mean ± SEM. (K–N) Examples of WT and PLPPR3 −/− neurons, and PLPPR3 −/− neurons expressing PLPPR3 ICDm or PLPPR3 ICDm S351A. Neurons were stained with anti-VGLUT1 and anti-BASP1 antibodies. Scale bars, 5 μm.

Article Snippet: Mouse: Primary hippocampal and cortical neurons (C57 Bl/6NCrl ) , The Jackson Laboratory , Cat#000664; RRID: IMSR_JAX:000664.

Techniques: Phospho-proteomics, Western Blot, Microscopy, Staining, Over Expression, Transfection, Recombinant, Expressing