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Figure Lengend Snippet: A-B Two weeks after TX or vehicle treatment, an IHKA injection was performed in WT/oil, WT/TX, and nSTAT3KO mice (n = 3 mice/group). Mice were killed 48 h after IHKA injection. Fluoro-Jade C (FJC) staining was performed in coronal sections (14-μm, ~8 sections per animal) obtained from all groups. A 1-in-10 series of sections from each brain was processed. Representative images of FJC-stained CA1, CA3, and hilus subregions ipsilateral to the IHKA injection are shown (A). Images were taken at 10X magnification. FJC-positive cells (positively stained cells per mm2) were counted blindly within standardized areas of the CA1, CA3, and hilar regions of the hippocampus ipsi- and contra-lateral to IHKA or IHSaline injection using ImageJ Analysis software (v. 1.53a). Number of FJC-positive cells in CA1, CA3, or hilus are reported as mean ± SEM (B). The mean number of positive cells in each region was calculated from all sections processed for each animal. No statistically significant differences were detected among groups in any of the 3 hippocampal regions tested. Statistical analysis was performed using a 1-way ANOVA, followed by Tukey’s post-hoc test. C-F. Immunofluorescence staining of NeuN (C, D) and VGLUT1 (E, F) was performed in coronal sections (5/mouse, ~1 in 10 series, 5 mice/group) from TX-treated WT and nSTAT3KO mice 5–6 weeks after IHSaline (WT, nSTAT3KO) or IHKA injection (WT+IHKA, nSTAT3KO+IHKA). Representative images of NeuN- and VGLUT1- stained CA1 and CA3 subregions ipsilateral to injection are shown in panels C and E. (contralateral staining shown in Fig S3). Images were acquired using 10X objective on a Nikon Eclipse TE2000-U fluorescence microscope. NeuN-positive cells were manually counted using ImageJ software. Statistically significant reductions in NeuN-positive (D) and VGLUT-positive (F) cells were observed in ipsilateral CA1 and CA3 following IHKA injection (compared to respective saline-treatment) in both WT and nSTAT3KO mice. No significant difference in CA1 or CA3 NeuN-positive or VGLUT-positive cells was detected between WT-IHKA and nSTAT3KO-IHKA. Statistical analysis was performed using 1-way ANOVA followed by Tukey’s post-hoc analysis. *p<0.05 and ****p<0.0001. G-H. Brains from TX-treated WT and nSTAT3KO mice were collected 5–6 weeks after IHSaline (controls) or IHKA injections, fixed by perfusion with 4% PFA and sectioned (14-μm coronal). Sections were stained with anti-ZnT3 antibody (~5 sections/mouse, 3 mice/group). Images of ZnT3 staining in the inner molecular layer (IML) above the upper blade of DGCs and ipsilateral to IHSaline or IHKA injection were obtained with a Nikon Eclipse TE2000-U fluorescence microscope at 10X magnification (G). Fluorescence intensity was quantified with ImageJ on 16 bit-converted images. The white squares demarcate the area used for the quantification of ZnT3 immunoreactivity. The area of ZnT3 immunoreactivity was quantified in 3 sections and values averaged to determine the levels of “sprouting” detected in each individual mouse (H). Mice injected with saline showed no labeling in the IML while the IHKA-injected mice showed presence of ZnT3 fluorescent staining indicative of mossy fiber sprouting. No statistically significant difference was observed in ZnT3 staining intensity between IHKA-WT (n= 3) and IHKA-nSTAT3KO (n= 3) mice (ANOVA, followed by Tukey’s post-hoc test).

Article Snippet: Slides dried 20 min at RT, PBS washed 3×5 min and antigen retrieval performed in sodium citrate buffer (10 mM Na 3 C 6 H 5 O 7 , 0.05%Tween-20; 10 min@95°C then 30 min@RT), followed by 3-PBS washes and blocking with 10% normal goat serum (NGS;Vector Labs) in PBST (0.3% Tween 100/PBS) for 1 h. Slides incubated overnight in anti-NeuN (#MAB377, Millipore; 1:600), anti-VGLUT1 (#AGC-035, Alomone; 1:400), or anti-ZnT3 (#197–002, Synaptic Systems; 1:500) primary antibody in 7.5%-NGS/0.3%-PBST, washed 3×5 min in PBS, incubated in AlexaFluor568 goat-anti-mouse (#A11031, Invitrogen; 1:800) or AlexaFluor488 goat-anti-rabbit (#A11034, Invitrogen; 1:600) secondary antibody for 1 h, washed in PBS 3×5 min, and mounted using Vectashield media/DAPI (#H1200, Vector Labs).

Techniques: Injection, Staining, Software, Immunofluorescence, Fluorescence, Microscopy, Saline, Labeling

Journal: bioRxiv

Article Title: Microglia undergo transcriptional, translational and functional adaptations to dark and light phases in laboratory mice

doi: 10.1101/2023.11.17.567571

Figure Lengend Snippet: Microglia display increased synaptic pruning during the active phase In the adult hippocampus. A Schematic representation of the experimental setup. Whole hippocampi were retrieved from perfused adult male mice either 4h post lights-off (active phase) or 4h post lights-on (sleep phase) for CD11b cell enrichment and subsequent flow cytometry analysis of vGlut1-posive cells. B Heatmap showing the differential expression (in average transcripts per million, TPM), for selected genes associated with microglial phagocytic functions from the total-RNA-seq of microglia 4h post lights-off or 4h post lights-on ( , 1 a.m. versus 1 p.m., Only genes that were significantly differentially expressed are represented, adj. p -value < 0.05) C Scatter plot showing the difference in percentage of vGlut1-positive microglia cells between the active and sleep phase, 4h post lights-off and on respectively, (unpaired t-test, t(5.84) = 4.84, p = 0.0031, N = 6/group). D Histogram showing the microglial vGlut1-PE mean fluorescence intensity normalized to spleen cells as negative control (lower panel). E Scatter plot showing the area under the curve (Au) for the microglial vGlut1 mean fluorescence intensity during the active (4h after light-off) and sleep (4h post light-on) phases, normalized to spleen cells as negative controls (unpaired t-test, t(10) = 2.98, p = 0.014, N = 6/group). F Heatmap showing differential expression (in average transcripts per million, TPM), for selected genes coding for chemokines (from the total-RNA-seq of microglia 1 p.m. versus 1 a.m.). Only genes that were significantly differentially expressed are represented (adj. p -value < 0.05). G Scatter plot comparing the percentage of CD45 hi cell population in the hippocampi of adult mice 4h after light-off (N = 17) and 4h post light-on (N = 13, unpaired t-test, t(28) = 3.08, p = 0.0046). Error bars represent the mean ± standard deviation. * p < 0.05, ** p < 0.01. Images created with Biorender.com.

Article Snippet: Following fixation, intracellular staining for vGLUT1 (1/200, Miltenyi Biotec, #130-120-764, 1h in 1x BD Permeabilization Buffer) was immediately performed.

Techniques: Flow Cytometry, Quantitative Proteomics, RNA Sequencing, Fluorescence, Negative Control, Standard Deviation

Journal: bioRxiv

Article Title: Microglia undergo transcriptional, translational and functional adaptations to dark and light phases in laboratory mice

doi: 10.1101/2023.11.17.567571

Figure Lengend Snippet: Gating strategy for the flow cytometry analysis of vGLUT1- positive inclusions within hippocampal microglia cells sorted during wither the active or the sleep phase. Supplementary Figure S6: A -log(FDR) values for biological processes associated with the genes deregulated in response to LPS in function of time of injection. B Unbiased Tmod enrichment analysis for reactome gene sets for the LPS response genes regulated by time of injection.

Article Snippet: Following fixation, intracellular staining for vGLUT1 (1/200, Miltenyi Biotec, #130-120-764, 1h in 1x BD Permeabilization Buffer) was immediately performed.

Techniques: Flow Cytometry, Injection

Journal: Experimental Neurobiology

Article Title: Sensory Stimulation-dependent Npas4 Expression in the Olfactory Bulb during Early Postnatal Development

doi: 10.5607/en23037

Figure Lengend Snippet: Information on primary antibodies used in western blot

Article Snippet: Anti- VGLUT1 , Atlas antibodies , Amab91041 , WB 1:1000.

Techniques: Western Blot, Concentration Assay

Journal: Experimental Neurobiology

Article Title: Sensory Stimulation-dependent Npas4 Expression in the Olfactory Bulb during Early Postnatal Development

doi: 10.5607/en23037

Figure Lengend Snippet: Effect of unilateral naris occlusion on OB interneurons during postnatal olfactory bulb development. (A) Immunoblot of GAD67 and VGLUT1 in P14 olfactory bulbs isolated from the open and closed sides of the naris. (B) The relative levels of GAD67 and VGLUT1 normalized by β-actin level (n=11, ***p<0.001). (C) Immunoblots of tyrosine hydroxylase (TH) and calretinin (CALR) normalized by β-actin level in P14 olfactory bulbs isolated from the open and closed sides of the naris. (D) The relative levels of TH and CALR were normalized by β-actin level (n=11, **p<0.01, ****p<0.0001).

Article Snippet: Anti- VGLUT1 , Atlas antibodies , Amab91041 , WB 1:1000.

Techniques: Western Blot, Isolation

Journal: Experimental neurology

Article Title: Dual Motor Cortex and Spinal Cord Neuromodulation Improves Rehabilitation Efficacy and Restores Skilled Locomotor Function in a Rat Cervical Contusion Injury Model

doi: 10.1016/j.expneurol.2021.113715

Figure Lengend Snippet: Proprioceptive afferent terminations on CTB-labelled ECR motoneurons. A, B) Confocal micrographs of VGlut1 on motoneurons. A: Rehab-only, B: Stim+rehab. Rehab-only group has more VGlut-1 terminations contacting the boarder and proximal dendrites of ECR motoneurons. VGlut1 on motoneurons is a marker for PA synaptic terminals. Figures illustrate greater monosynaptic PA-motoneuron connections after rehab-alone, whereas stim+rehab seems to attenuate such synaptic formation. Scale bars: 50 μm. C) Bar graph showing rehab-only group has significantly more VGlut-1 per motoneurons (boarder and proximal dendrites) compared to stim+rehab.

Article Snippet: Vesicular glutamate transporter 1 (VGlut1) was localized using a primary anti-VGlut1 antibody (1:10; NeuroMab) and a secondary donkey anti-mouse conjugated to Cy5.

Techniques: Marker