Journal: eLife

Article Title: Sparse genetically defined neurons refine the canonical role of periaqueductal gray columnar organization

doi: 10.7554/eLife.77115

Figure Lengend Snippet: ( A ) Example histology images showing immunostaining of pan-neuronal marker NeuN (top row), viral-mediated expression of GFP in CCK-expressing cells (middle row), and overlay of NeuN and CCK-GFP (bottom row) in the dorsolateral (left column), lateral (middle column), and ventrolateral (right column) PAG. Scale bars, 10 μm. ( B ) Raw counts of CCK-GFP+ and NeuN+ cells in the dorsolateral PAG (dlPAG), lPAG, and vlPAG. ( C ) Fraction of NeuN-labeled cells that are also GFP-labeled in the dlPAG and l/vlPAG. CCK-expressing cells comprise ~5% of l/vlPAG neurons and constitute significantly more of l/vlPAG neurons than dlPAG neurons (n = 4; paired t-test, ** P =.0032). ( D ) Immunostaining of glutamatergic marker vGlut2 in CCK cells. Example histology images showing vGlut2 (top), CCK-GFP (middle) and vGlut2/GFP overlay (bottom). White arrow indicates vGlut2+/GFP+ cell. Dashed outline indicates vGlut2+/GFP- cell. Scale bar, 10 μm. ( E ) 9.6% of vGlut2-labeled cells in the l/vlPAG are also GFP-labeled (n = 4; 302 vGlut2/GFP+ of 3115 vGlut2+ cells). ( F ) A majority (94.8%) of GFP-labeled cells in the l/vlPAG are also vGlut2-labeled (n = 4; 302 vGlut2+/GFP+ of 317 GFP+ cells). Errorbars: mean ± SEM.

Article Snippet: Sections were then incubated at 4°C for 16 hr with polyclonal anti-VGLUT2 antibody (#AGC-036, Alomone Labs) made in rabbit (1:500 dilution) in blocking solution.

Techniques: Immunostaining, Marker, Expressing, Labeling

Journal: eLife

Article Title: Sparse genetically defined neurons refine the canonical role of periaqueductal gray columnar organization

doi: 10.7554/eLife.77115

Figure Lengend Snippet: ( A ) In situ hybridization labeling of l/vlPAG neurons showing mRNAs for CCK (green) and VGLUT2 (red). Nuclei stained by DAPI are shown in blue. Example image (×40 objective) of vglut2-labeled cells (red rectangle) and double-labeled vglut2/CCK-labeled (green rectangle) in l/vlPAG. Scale bar, 20 µm. ( B ) Zoomed-in images of subregions in ( A ). Scale bar, 20 µm. ( C ) 8.58% of vglut2-labeled cells are also CCK-labeled (15 of 183 total cells). Errorbars: mean ± SEM.

Article Snippet: Sections were then incubated at 4°C for 16 hr with polyclonal anti-VGLUT2 antibody (#AGC-036, Alomone Labs) made in rabbit (1:500 dilution) in blocking solution.

Techniques: In Situ Hybridization, Labeling, Staining

Journal: eNeuro

Article Title: nNOS-Expressing Neurons in the Ventral Tegmental Area and Substantia Nigra Pars Compacta

doi: 10.1523/ENEURO.0381-18.2018

Figure Lengend Snippet: Primary antibodies

Article Snippet: Anti-VGLUT2 , Rabbit , Alomone (AGC036; AB_2340950 ) , 1:500.

Techniques: Concentration Assay

Journal: Nature Communications

Article Title: Sema7A/PlxnCl signaling triggers activity-dependent olfactory synapse formation

doi: 10.1038/s41467-018-04239-z

Figure Lengend Snippet: Synapse formation and dendrite maturation in the Sema7A and PlxnC1 KOs. a Synapse markers within glomeruli in the Sema7A KO. OB sections of the WT and Sema7A KO at P5 and 8w were immunostained with antibodies against vGlut2 and GluR1, pre- and post-synaptic markers, respectively. Staining photos are shown for P5 sections. Signal intensities within the rI7 glomeruli were normalized by the pre-synaptic signals in the ONL and post synaptic signals in the EPL. Fluorescent signals of GL/ONL and GL/EPL in glomeruli are compared between the WT and Sema7A KO. Scale bars=25 μm. * p < 0.05, ** p < 0.01 (Student’s t -test). Error bars indicate SD ( n = 5, 6, 2, 3 animals). b Dendrite morphology of M/T cells in the Sema7A KO. EYFP-tagged rI7 glomeruli (green) were detected by immunostaining with anti-GFP antibodies. M/T cells (yellow) were visualized by Lucifer yellow (LY) injection into the rI7 glomeruli of the WT and Sema7A KO at P5 (left). The numbers of M/T cells with one dendrite (mature) and those with multiple dendrites (immature) to GL were counted for the rI7 glomeruli. The ratios (%) of mature (blue) and immature (white) M/T cells are shown (right): WT, 26/34 (76.5 %); KO, 8/34 cells (23.5 %). n = 20 glomeruli. Scale bar=10 μm. c Synapse markers within glomeruli in the PlxnC1 cKO. OB sections of the WT and PlxnC1 cKO at P5 and 8w were analyzed for synapse markers. Staining photos are shown for P5 sections. Signal intensities within the rI7 glomeruli were shown. Scale bars=25 μm. * p < 0.05, ** p < 0.01 (Student’s t -test). Error bars indicate SD ( n = 5, 5, 2, 3 animals). d Dendrite morphology in the PlxnC1 cKO. M/T cells (yellow) were visualized by LY injection into the rI7 glomeruli of the WT and PlxnC1 cKO at P5 (left). The ratios (%) of mature (blue) and immature (white) M/T cells are shown (right): WT, 24/31 (77.4 %); KO, 7/31 (22.6 %). n = 23 glomeruli. Scale bar=20 μm

Article Snippet: Antibodies against Sema7A (goat, 1:3000, #AF-1835), PlxnC1 (goat, 1:3000, discontinued), and CNG-A2 (rabbit, 1:200, #APC-045), vGlut2 (guinea pig, 1:1000, #AB2251-l), GluR1 (rabbit, 1:1000, #ab51092), GFP (rabbit, 1:1000, #A-10260), Lucifer yellow (rabbit, 1:2000, #A-5750), and OMP (goat, 1:2000, #Cat. No. 019-22291), were purchased from R&D Systems, Abcam, Alomone Labs, Millipore, Invitrogen, Abcam, Thermo Fisher Scientific, Thermo Fisher Scientific, and Wako Chemicals, respectively.

Techniques: Staining, Immunostaining, Injection

Journal: Nature Communications

Article Title: Sema7A/PlxnCl signaling triggers activity-dependent olfactory synapse formation

doi: 10.1038/s41467-018-04239-z

Figure Lengend Snippet: Sema7A promotes dendrite maturation and synapse formation. Left, Sema7A levels in the MOR29B and MOR29A glomeruli. OB sections at P4 were immunostained with anti-Sema7A antibodies, and signal intensities of Sema7A are compared (see also Fig. ). * p < 0.05 (Student’s t -test). Error bars indicate standard deviation SD ( n = 8 glomeruli). Middle, Pre- and post-synaptic markers in the MOR29A and MOR29B glomeruli. OB sections at P4 were also immunostained with antibodies against vGlut2 and GluR1, pre- and post-synaptic markers, respectively. Relative fluorescent signals (MOR29B/MOR29A glomeruli) are compared. * p < 0.05 (Student’s t -test). Error bars indicate standard deviation SD ( n = 8 glomeruli). Right, Dendrite maturation of M/T cells in the MOR29A and MOR29B glomeruli. M/T-cell dendrites were visualized by LY injection into the MOR29A and MOR29B glomeruli at P4. The ratios (%) of mature and immature M/T cells in the MOR29A and MOR29B glomeruli are shown: MOR29A, 8/29 (27.6 %); MOR29B, 22/28 (78.6%). n = 16 glomeruli

Article Snippet: Antibodies against Sema7A (goat, 1:3000, #AF-1835), PlxnC1 (goat, 1:3000, discontinued), and CNG-A2 (rabbit, 1:200, #APC-045), vGlut2 (guinea pig, 1:1000, #AB2251-l), GluR1 (rabbit, 1:1000, #ab51092), GFP (rabbit, 1:1000, #A-10260), Lucifer yellow (rabbit, 1:2000, #A-5750), and OMP (goat, 1:2000, #Cat. No. 019-22291), were purchased from R&D Systems, Abcam, Alomone Labs, Millipore, Invitrogen, Abcam, Thermo Fisher Scientific, Thermo Fisher Scientific, and Wako Chemicals, respectively.

Techniques: Standard Deviation, Injection