Journal: eLife

Article Title: Presynaptic LRP4 promotes synapse number and function of excitatory CNS neurons

doi: 10.7554/eLife.27347

Figure Lengend Snippet: ( A ) Domain structure of Drosophila LRP4. Numbers indicate amino acids. EXT, extracellular side. INT, intracellular side. ( B ) Representative confocal image stack of a control Drosophila brain stained with antibodies against endogenous LRP4 (green) and Bruchpilot (inset, magenta) demonstrating expression throughout the brain. ( C ) Representative confocal image stack of an lrp4 dalek null brain stained with antibodies against LRP4 (green) and Brp (inset, magenta) demonstrating antibody specificity. ( D ) Representative confocal image of a Drosophila brain expressing UAS-Syt-HA via lrp4-GAL4 and stained with antibodies to HA ( D , green) and N-Cadherin (inset, magenta). The expression pattern resembles that of endogenous LRP4, supporting the specificity of lrp4-GAL4 . ( E ) Representative single slice within a single antennal lobe glomerulus of a brain processed for expansion microscopy (proExM) expressing LRP4-HA and Brp-Short-mStraw in all ORNs via pebbled-GAL4 and stained with antibodies to HA ( E , E” , green) and mStraw ( E’ - E” , magenta). LRP4 localizes to synaptic neuropil regions. ( F ) High magnification image of the region bounded by dashed lines in ( E ) and stained as above. Arrows indicate LRP4-HA localization adjacent to / not directly overlapping with Bruchpilot-Short. Arrowheads indicate overlapping LRP4-HA and Brp-Short localization. ( G–K ) Representative high magnification confocal stack images of neuronal cell bodies surrounding the antennal lobe in animals expressing UAS-mCD8-GFP via lrp4-GAL4 and stained for antibodies against GFP (G-K, green) and other cell-type markers ( G’ - K’ , magenta). Merge channels ( G’’–K’’ ) show colocalization of lrp4 with the neuronal marker ELAV ( G’’ ) but not the glial cell marker Repo ( H’’ ). Neurons positive for lrp4 show colocalization with choline acetyltransferase (ChAT, I’’ ), and the vesicular glutamate transporter (vGlut, J’’ ), but little to no colocalization with the inhibitory neurotransmitter GABA ( K’’ ), suggesting that lrp4 -positive cells are largely excitatory neurons. The percentage of GFP-positive cells that are ALSO positive for the cell-type specific marker are as follows: Elav = 99.50 ± 0.19% overlap; Repo = 0.38 ± 0.18% overlap; ChAT = 59.13 ± 2.48% overlap; vGlut = 22.38 ± 1.28% overlap; GABA = 0.25 ± 0.16% overlap. For all cases, n = 8 animals, ≥ 200 cells per animal. Values = mean ± s.e.m. Scale bars = 50 µm ( B–D ), 150 μm (B-D, insets), 25 μm ( E–F ), 10 μm ( G–K ). DOI: http://dx.doi.org/10.7554/eLife.27347.003

Article Snippet: The following primary antibodies were used: mouse antibody to Bruchpilot (1:40, DSHB, Catalog #mAbnc82, Iowa City, IA) , rabbit antibody to Synaptotagmin I (1:4000) , rat antibody to N-Cadherin (1:40, DSHB, Catalog #mAbDN-EX #8, Iowa City, IA) , rat antibody to HA (1:100, Roche, Catalog #11867423001, Basel, Switzerland), mouse antibody to choline acetyltransferase (ChAT) (1:100, DSHB, Catalog #mAbChAT4B1, Iowa City, IA) , mouse antibody to ELAV (DSHB, mAb9F8A9, 1:100) , rabbit antibody to GABA (1:200, Sigma-Aldrich, Catalog #A2052, St. Louis, MO), mouse antibody to Repo (1:100, DSHB, Catalog #mAb8D12, Iowa City, IA) , rabbit antibody to vGlut (1:500) , rabbit antibody to dsRed (1:250, Clontech, Catalog #632496, Mountain View, CA), chicken antibody to GFP (1:1000, Aves Labs, Catalog #GFP-1020, Tigard, OR), Alexa647-conjugated goat antibody to HRP (1:100, Jackson ImmunoResearch, Catalog #123-605-021, West Grove, PA).

Techniques: Control, Staining, Expressing, Microscopy, Marker

Journal: International journal of molecular sciences

Article Title: Effectiveness of Combinational Treatments for Alzheimer's Disease with Human Neural Stem Cells and Microglial Cells Over-Expressing Functional Genes.

doi: 10.3390/ijms24119561

Figure Lengend Snippet: Figure 1. Expression of mRNAs and production of functional proteins in human neural stem cells and microglial cells, and amyloid-β (Aβ) clearance by microglial cells. (A) RT-PCR analysis of ChAT, NEP and SRA mRNAs. (B) Immunocytochemical staining on ChAT, NEP and SRA (green). (C) Clearance of Aβ peptides by HMO6 (#), HMO6.NEP (•) and HMO6.SRA (▼). * Significantly different from HMO6 cells at each time point (p < 0.05).

Article Snippet: Immunohistochemical Analysis of hMito and Functional Proteins The mouse brains were perfusion-fixed with 4% paraformaldehyde solution and postfixed for 48 h, followed by cryoprotection in 30% sucrose for 72 h. Coronal cryosections in 30-μm thickness were prepared and processed for double immunostaining of human mitochondria (hMito) and/or ChAT, NEP or SRA using antibodies specific for hMito (1:100, Millipore), ChAT (1:100, Invitrogen), NEP (1:100, Invitrogen) and SRA (1:100, Bioss).

Techniques: Expressing, Functional Assay, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: International journal of molecular sciences

Article Title: Effectiveness of Combinational Treatments for Alzheimer's Disease with Human Neural Stem Cells and Microglial Cells Over-Expressing Functional Genes.

doi: 10.3390/ijms24119561

Figure Lengend Snippet: Figure 4. Recovery of learning and memory functions four weeks post-injection in passive avoid- ance (A) Morris water-maze (B) studies, and tracking of water-maze swimming performance (C). #: Normal, •: AF64A alone, ▼: F3, △: F3.ChAT, ■: HMO6, □: HMO6.NEP, ◆: HMO6.SRA, 3: F3.ChAT + HMO6.NEP, ▲: F3.ChAT + HMO6.SRA. * Significantly different from normal (p < 0.05). # Significantly different from AF64A alone (p < 0.05).

Article Snippet: Immunohistochemical Analysis of hMito and Functional Proteins The mouse brains were perfusion-fixed with 4% paraformaldehyde solution and postfixed for 48 h, followed by cryoprotection in 30% sucrose for 72 h. Coronal cryosections in 30-μm thickness were prepared and processed for double immunostaining of human mitochondria (hMito) and/or ChAT, NEP or SRA using antibodies specific for hMito (1:100, Millipore), ChAT (1:100, Invitrogen), NEP (1:100, Invitrogen) and SRA (1:100, Bioss).

Techniques: Injection

Journal: International journal of molecular sciences

Article Title: Effectiveness of Combinational Treatments for Alzheimer's Disease with Human Neural Stem Cells and Microglial Cells Over-Expressing Functional Genes.

doi: 10.3390/ijms24119561

Figure Lengend Snippet: Figure 5. Immunohistochemical identification of human neural stem cells (F3) and microglial cells (HMO6) and functional proteins (ChAT, NEP and SRA) via double immunostaining for human mitochondria (hMito) and ChAT, NEP or SRA.

Article Snippet: Immunohistochemical Analysis of hMito and Functional Proteins The mouse brains were perfusion-fixed with 4% paraformaldehyde solution and postfixed for 48 h, followed by cryoprotection in 30% sucrose for 72 h. Coronal cryosections in 30-μm thickness were prepared and processed for double immunostaining of human mitochondria (hMito) and/or ChAT, NEP or SRA using antibodies specific for hMito (1:100, Millipore), ChAT (1:100, Invitrogen), NEP (1:100, Invitrogen) and SRA (1:100, Bioss).

Techniques: Immunohistochemical staining, Functional Assay, Double Immunostaining