mouse anti vglut1  (NeuroMab)

Journal: bioRxiv

Article Title: Tau oligomers modulate synapse fate by eliciting progressive bipartite synapse dysregulation and synapse loss

doi: 10.1101/2025.09.19.677215

Figure Lengend Snippet: A Representative confocal images of immunolabeling of surface GluA1-containing AMPARs (red) and vGluT1 (green), a synaptic marker, in human neurons. Neurons were treated with tau monomers or oligomers for 30 min and then they were either unstimulated or stimulated with chemical induction of LTP (cLTP). The neurons were fixed at approximately 30 min after cLTP induction. Scale bar: 5 μm. B Quantification of the intensity of surface GluA1 at synapses normalized to unstimulated neurons treated with tau monomers (n = 12 neurons/group; * p < 0.05 Unpaired Student’s t -test). C Workflow of protocol to biotinylate postsynaptic proteins in close proximity with APEX2-PSD-95 in human neurons, to pulldown the biotinylated proteins using streptavidin beads and to identify the biotinylated peptides using mass spectrometry. D Immunoblot of flag-tagged APEX2-PSD-95, biotinylated proteins (streptavidin), and GAPDH loading control from human neurons with or without APEX2-PSD-5 expression and with or without H 2 O 2 treatment for 1 min. E Human neurons expressing APEX2-PSD-95 incubated with biotin-phenol for 30 min and stimulated with H 2 O 2 for 1 min. Cells were fixed and immunostained for APEX2-PSD-95 (flag, red), biotinylated proteins (Streptavidin, blue) and a synaptic marker (Synapsin, green). Scale bar: 5 μm. F Workflow of treatment of human neurons with tau oligomers for proximity labeling of postsynaptic proteins. G SynGO cellular component analyses of 545 biotinylated proteins identified by mass spectrometry from neurons that were untreated or exposed to tau oligomers. Size of circles denotes protein count, colors denote p-values and protein ratio denotes protein count over total number of proteins. H SynGO analysis showed 130 total annotated postsynaptic proteins including the proteins classified in child terms. I Venn diagram analysis of the 545 biotinylated proteins detected in untreated neurons or in neurons 1 hour after tau oligomer exposure highlighting the significantly upregulated (red) and downregulated (blue) proteins in the tau oligomer-treated neurons compared to untreated neurons (n = 5-6 cultures/group; p < 0.05 with absolute average log 2 ratio > 0.25). J Gene Set Enrichment Analysis on significantly downregulated proteins in tau oligomer-treated neurons showing GO molecular function. K Analyses of the biotinylated proteins that were significantly downregulated in tau oligomer-exposed neurons. These proteins were also reduced in postmortem AD brain tissue . L Representative confocal images of immunostaining of Myosin-Va (red), PSD-95 (green) and vGluT1 (blue) in human neurons treat with tau oligomers for 30 min and fixed 1 hour later. Scale bar: 5 μm. M Quantification of the postsynaptic Myosin-Va immunolabeling intensity that colocalized with postsynaptic (PSD-95) and presynaptic (vGluT1) markers in human neurons treated with tau oligomers for 30 min and fixed after 1 hour (n = 15-16 images/group; ***, p < 0.001, Unpaired Student’s t -test).

Article Snippet: Primary antibodies used for western blot and immunocytochemistry were: Tau5 (AB_80579, Abcam), Streptavin HRP (S911, Invitrogen), GAPDH (MAB374, Sigma), Rabbit FLAG (F7425, Sigma), Mouse FLAG (F3165, Sigma), PSD-95 (MA1046, ThermoFisher), GluA2/3 (AB1506, MilliporeSigma), Cy3 Streptavidin (AB_2337244, Jackson Immunoresearch), Mouse vGluT1 (MAB5502, MilliporeSigma), Guinea Pig vGluT1 (AB5905, MilliporeSigma), GluA1 (ABN241, MilliporeSigma), GluN1 (114 011, SynapticSystems), Synapsin (5297S, Cell Signaling), Rabbit MAP2 (4542S, Cell Signaling), Chicken MAP2 (NB300 213, Novus Biologicals), NeuN (AB177487, Abcam), GFAP (PA110004, Invitrogen) Secondary antibodies used were: Anti-mouse Alexa Fluor 488 (115-545-166, Jackson Immunoresearch), Anti-guinea pig Alexa Fluor 546 (A-11074, Life Technologies), Anti-mouse Alexa Fluor 546 (A-11030, Invitrogen), Anti-rabbit Alexa Fluor 647 (111-605-144, Jackson Immunoresearch), Anti-chicken Alexa Fluor 647 (A32933, Invitrogen)

Techniques: Immunolabeling, Marker, Mass Spectrometry, Western Blot, Control, Expressing, Incubation, Labeling, Immunostaining

Journal: bioRxiv

Article Title: Tau oligomers modulate synapse fate by eliciting progressive bipartite synapse dysregulation and synapse loss

doi: 10.1101/2025.09.19.677215

Figure Lengend Snippet: Representative confocal images of immunostaining for the total levels of GluA2/3 subunit-containing AMPARs (red), including both surface and intracellular AMPAR pools, and PSD-95 (green) in human neurons at ( A ) 7 or ( D ) 14 days after the acute exposure to tau oligomers and vehicle-treated control neurons. The mean intensity of GluA2/3 immunofluorescence that colocalized with PSD-95 puncta was quantified (arrowheads) at ( B ) 7 or ( E ) 14 days post-tau oligomer exposure and normalized to vehicle-treated control neurons. The mean intensity of PSD-95 puncta was also quantified at ( C ) 7 or ( F ) 14 days post-tau oligomer exposure normalized to vehicle-treated controls (n = 12-24 images/group; * p < 0.05, *** p < 0.05, Unpaired Student’s t -test). G Representative confocal images of surface GluA1-containing AMPARs labeled with a GluA1 antibody (red) that co-localized with vGluT1 (green), a synaptic marker, in human neurons (arrowheads). Neurons were treated with vehicle or tau oligomers for 30 min, and then 14 days later, the neurons were either unstimulated or stimulated by cLTP induction. Scale bar: 5 μm. H Graph of the immunolabeling intensity of surface GluA1 quantified at synapses 14 days after tau oligomer treatment. The surface GluA1 intensities across groups were normalized to unstimulated control neurons (n = 12 neurons/group; * p < 0.05 Unpaired Student’s t -test).

Article Snippet: Primary antibodies used for western blot and immunocytochemistry were: Tau5 (AB_80579, Abcam), Streptavin HRP (S911, Invitrogen), GAPDH (MAB374, Sigma), Rabbit FLAG (F7425, Sigma), Mouse FLAG (F3165, Sigma), PSD-95 (MA1046, ThermoFisher), GluA2/3 (AB1506, MilliporeSigma), Cy3 Streptavidin (AB_2337244, Jackson Immunoresearch), Mouse vGluT1 (MAB5502, MilliporeSigma), Guinea Pig vGluT1 (AB5905, MilliporeSigma), GluA1 (ABN241, MilliporeSigma), GluN1 (114 011, SynapticSystems), Synapsin (5297S, Cell Signaling), Rabbit MAP2 (4542S, Cell Signaling), Chicken MAP2 (NB300 213, Novus Biologicals), NeuN (AB177487, Abcam), GFAP (PA110004, Invitrogen) Secondary antibodies used were: Anti-mouse Alexa Fluor 488 (115-545-166, Jackson Immunoresearch), Anti-guinea pig Alexa Fluor 546 (A-11074, Life Technologies), Anti-mouse Alexa Fluor 546 (A-11030, Invitrogen), Anti-rabbit Alexa Fluor 647 (111-605-144, Jackson Immunoresearch), Anti-chicken Alexa Fluor 647 (A32933, Invitrogen)

Techniques: Immunostaining, Control, Immunofluorescence, Labeling, Marker, Immunolabeling