Journal: The Journal of comparative neurology
Article Title: Cerebellar premotor output neurons collateralize to innervate the cerebellar cortex
doi: 10.1002/cne.23787
Figure Lengend Snippet: BDA- and virally- labeled boutons are immunopositive for SV2. A. (far left) A schematic diagram illustrating site of BDA injection to the ventrolateral thalamus (VL) is shown; (left) A cerebellar mossy fiber rosette located in the Crus I lobule labeled with BDA after a VL injection was immunopositive for SV2 (middle) as shown in the overlay (right; n=3). Confocal stack thickness was 1.35 μm. B–C. (far left) Schematic diagram of site of BDA injections into the granule cell layer. (See text for the range of injection sites for these experiments.) (left) Terminal boutons labeled with BDA in the red nucleus (RN) after a granule cell layer injection at the base of the Simple lobule near the primary fissure (n=5) expressed SV2 (middle) as shown in the overlay (right). Confocal stack thickness was 1.7 μm. C. (left) BDA-labeled terminal boutons were observed in VL following granule cell layer injection at the base of the Simple lobule near the primary fissure (n=5); SV2 immunostaining (middle) overlayed with labeled terminal (right). Confocal stack thickness was 0.34 μm. D–E. D. (far left) A schematic of AAV1-CAG-Flex-eGFP injection into the red nucleus of Ntsr1-Cre mice is shown. (left) Terminal boutons labeled with GFP were observed in the granule cell layer of the 4/5 lobule following virus injections into the RN of Ntsr1-Cre mice (n=5); these terminals expressed SV2 (middle) as shown in overlay (right). Confocal stack thickness was 1.89 μm. E. (left) GFP-expressing terminal boutons were seen in VL following viral injections to the RN of Ntsr1-Cre mice (n=5); SV2 immunostaining (middle) is overlayed with boutons (right). Confocal stack thickness was 1.62 μm. Scales, A,D = 5 μm; B,C and E = 10 μm.
Article Snippet: For immunolabeling, sections were blocked in a solution of 10% Normal Goat Serum (Invitrogen) in PB or in the permeabilization solution described above (1hr) and then incubated with one or two of the following primary antibodies (host in parenthesis): (mouse) Synaptic Vesicle Protein 2 (SV2; Developmental Studies Hybridoma Bank Cat# sv2, RRID:AB_528480; for 48 hours at 1:400; concentration determined from serial dilution test); (rabbit) anti-metabotropic Glutamate receptor 2+3 (mGluR 2/3; Abcam; Cat# ab6438, Lot# GR12051, RRID: AB_10307; manufacturer recommended dilution) for 24–48 hours at 1:500; (rabbit) anti-neurogranin for 24 hours at 1:500 (Millipore; Cat#AB5620, Lot# 2441899; RRID: AB_91937; manufacturer recommended dilution) ( ). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Immunogen Manufacturer, Catalog/Lot number, RRID, Host Species Concentration Used Synaptic Vesicle Protein 2 Transmembrane glycoprotein of ~95kDa on cytoplasmic side of synaptic vesicles Developmental Studies Hybridoma Bank, University of Iowa, Cat # sv2, RRID: AB_528480, mouse monoclonal 1:400 Anti-metabotropic Glutamate receptor 2+3 Peptide (NGREVVDSTTSSL) corresponding to 13 c-terminus amino acid of mGluR2 and 3 in rat Abcam, Cat# ab6438, Lot# GR12051, RRID: AB_10307, rabbit polyclonal 1:500 Anti-Neurogranin Recombinant rat neurogranin (complete sequence) Millipore, Cat# AB5620, Lot# 2441899, RRID: AB_91937, rabbit polyclonal 1:500 Anti-GFP GFP antibody Abcam, Cat# ab6556, Lot# GR154034-1, RRID: AB_305564, rabbit polyclonal 1:50 Open in a separate window Antibodies Used After three washes in PB, sections were then incubated with secondary antibodies at a 1:400 dilution (90 min).
Techniques: Labeling, Injection, Immunostaining, Virus, Expressing
