Journal: Medical research archives

Article Title: Identification of amacrine neurons with a glycinergic and GABAergic phenotype in the mouse retina

doi: 10.18103/mra.v10i1.2624

Figure Lengend Snippet: List of antibodies used in this study

Article Snippet: Anti-Mouse (CY3) , Donkey , Jackson Immunoresearch Laboratories, 715–165-150 , .

Techniques:

Journal: Medical research archives

Article Title: Identification of amacrine neurons with a glycinergic and GABAergic phenotype in the mouse retina

doi: 10.18103/mra.v10i1.2624

Figure Lengend Snippet: A) Vertical sections from the central retina of the pGlyT2-eGFP mouse were subjected to immunostaining and analyzed by confocal microscopy. Z-stack images were acquired and an optical section from the middle of the stack selected for presentation. Left panel: bright field showing the retinal layers; middle panel, eGFP expression; right panel, merge image. Scale bar, 20 μm. B) Vertical section stained with anti-Syntaxin 1 (HPC-1) antibodies and DAPI followed by CY-3 labeled secondary antibodies. C) Same as B but sections labeled with rabbit antibodies against GlyT1 or D) anti-glycine. Scale bar, 10 μm. Abbreviations: INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. E) Quantitation of EGFP-positive or EGFP/GlyT1 neurons. Cell counts were obtained from a minimum of 100 HPC-1 positive cells per retina, for a total of three retinas from three different mice. The plots represent the percent of HPC-1 positive cells +/− SE. Open arrowhead in C and D indicate eGFP labeled cells devoid of GlyT1or Glycine immunoreactivity, respectively.

Article Snippet: Anti-Mouse (CY3) , Donkey , Jackson Immunoresearch Laboratories, 715–165-150 , .

Techniques: Immunostaining, Confocal Microscopy, Expressing, Staining, Labeling, Quantitation Assay

Journal: Medical research archives

Article Title: Identification of amacrine neurons with a glycinergic and GABAergic phenotype in the mouse retina

doi: 10.18103/mra.v10i1.2624

Figure Lengend Snippet: A) Vertical sections from the central retina of the pGlyT2-eGFP mouse were immunolabeled with antibodies recognizing the GABA synthetizing enzyme GAD67 and rabbit GlyT1 antibodies or synaptic systems or followed by the corresponding secondary CY-3 and CY-5 labeled antibodies. Z-stack images were acquired through the corresponding channels and an optical section from the middle of the stack selected and used for this figure panels. B) Sections were stained as in A but using the rabbit GABA transporter anti-GAT1 and rat anti-GlyT1 antibodies or C) GAT3 and rat GlyT1. Scale bar 10 μm. Abbreviations: INL, inner nuclear layer; IPL, inner plexiform layer. Open arrowhead in A-C indicate eGFP labeled cells containing GlyT1 and GAD67, or GAT1 or GAT3. D) A minimum of 100 HPC-1 positive cells per retina from three different eyes were used to quantify the number of GAD67 or GlyT1 positive cells and those containing both markers. The plots represent the percent of GAD67 or/and GlyT1 positive cells +/− SE. E) Same as D but normalized by 100 EGFP positive ACs.

Article Snippet: Anti-Mouse (CY3) , Donkey , Jackson Immunoresearch Laboratories, 715–165-150 , .

Techniques: Immunolabeling, Labeling, Staining

Journal: Medical research archives

Article Title: Identification of amacrine neurons with a glycinergic and GABAergic phenotype in the mouse retina

doi: 10.18103/mra.v10i1.2624

Figure Lengend Snippet: Vertical section of the central mouse retina were labeled with antibodies against the protein gephyrin (upper panel) or the GlyR subunits using the mAb4a antibody that recognizes all the receptor subunits, followed by staining with CY-3 labeled secondary antibodies. Confocal Z-stack images were acquired and an optical section from the middle of the stack selected and used for presentation. Scale bar, 10 μm.

Article Snippet: Anti-Mouse (CY3) , Donkey , Jackson Immunoresearch Laboratories, 715–165-150 , .

Techniques: Labeling, Staining

Journal: Medical research archives

Article Title: Identification of amacrine neurons with a glycinergic and GABAergic phenotype in the mouse retina

doi: 10.18103/mra.v10i1.2624

Figure Lengend Snippet: A) Vertical sections from the central retina of C57BL/6J mice were subjected to immunostaining with GlyT1 and GAD67 antibodies followed by incubation with CY-3 and Alexa-488 labeled secondary antibodies, respectively. Scale bar, 50 μm. A1 and A2) Magnification of the areas enclosed in panel A, cells labeled for GAD67 and GlyT1 are marked by an arrow. B) Vertical sections were stained with HPC-1, GlyT1 and GAD67 and the resulting images used for quantitation as described in ​in4D.4D. C) Vertical sections from the central retina of the transgenic line pGAD67-EGFP were labeled with GlyT1 antibody from Synaptic Systems followed by incubation with CY-3 labeled secondary antibodies. Nuclei were stained with DAPI. Arrows indicate co-expression of GlyT1 and eGFP in amacrine neurons. Scale bar, 10 μm for B and C. D) Sections from the pGAD67-EGFP were subjected to quantitation analysis, as described in ​in4D.4D. Scale bar, 10 μm.

Article Snippet: Anti-Mouse (CY3) , Donkey , Jackson Immunoresearch Laboratories, 715–165-150 , .

Techniques: Immunostaining, Incubation, Labeling, Staining, Quantitation Assay, Transgenic Assay, Expressing

Journal: Medical research archives

Article Title: Identification of amacrine neurons with a glycinergic and GABAergic phenotype in the mouse retina

doi: 10.18103/mra.v10i1.2624

Figure Lengend Snippet: A) Primary dissociated retinal cultures were fixed with paraformaldehyde and incubated with antibodies recognizing GAD67 and GlyT1 followed by staining with the corresponding CY-3 and CY-5 labeled antibodies, respectively. The inhibitory neurons were heterogeneous and contained eGFP and GlyT1 (long arrows), or eGFP, GAD67 and GlyT1 (open arrowheads), or GAD67 (thin open arrows). B) Cells were subjected to immunostaining with antibodies against GAT1 and GlyT1 or C) against GAT3 and GlyT1 as described under Experimental Procedures. Scale bars, 10 μm.

Article Snippet: Anti-Mouse (CY3) , Donkey , Jackson Immunoresearch Laboratories, 715–165-150 , .

Techniques: Incubation, Staining, Labeling, Immunostaining

Journal:

Article Title: Brain Lipid Binding Protein in Axon-Schwann Cell Interactions and Peripheral Nerve Tumorigenesis

doi: 10.1128/MCB.23.6.2213-2224.2003

Figure Lengend Snippet: mRNA expression analysis in Nf1 mutant mouse Schwann cells. (A) Microarray analysis was used to compare genome-wide expression levels between normal mouse Schwann cells and Nf1 mutant Schwann cells. The control for each comparison was Cy3-labeled cDNA generated from normal mouse Schwann cell mRNA. For each of four Nf1 mutant Schwann cell samples (Nf1+/−, Nf1−/−, Nf1−/− TXF, and Nf1−/− TXF treated with FTI), mRNA was used as a template to synthesize Cy5-labeled cDNA. Cy3- and Cy5-labeled cDNA probes were hybridized simultaneously to the Incyte Genomics MouseGEM 1.0 cDNA microarray. Relative intensities of Cy3 versus Cy5 fluorescent signals for each cDNA target sequence were analyzed with GeneSpring software. The most changes were observed in the Nf1−/− TXF cells (genes upregulated in Nf1−/− TXF are red; genes downregulated in Nf1−/− TXF are green). Expression of one target cDNA, BLBP (black line), was 26-fold above normal in the Nf1−/− TXF cells and not normalized by FTI treatment. (B) RT-PCR analysis confirmedthe microarray result of elevated BLBP expression in Nf1−/− TXF cells. Reverse transcriptase (RT) was omitted from duplicate samples to control for DNA contamination. Primers for BLBP (∼200-bp amplicon) and actin control primers (∼500-bp amplicon) were included in the mixture for each 40-cycle reaction. The plasmid positive control for BLBP amplification is the UniGEM clone (Incyte Genomics) containing the BLBP cDNA insert spotted on the microarray. (C) Quantitative real-time PCR of BLBP normalized to GAPDH resulted in a 145-fold change over expression in Nf1−/− TXF cells compared to wild-type mouse Schwann cells. Rn, fluorescent signal intensity; horizontal starred line, chosen threshold at geometric phase of amplification.

Article Snippet: In DRGN cocultures, neurofilament was visualized by incubation with anti-NF-15g1 antibodies ( 38 ) diluted 1:5, followed by donkey anti-mouse Cy3-labeled secondary antibodies (Jackson Immunoresearch).

Techniques: Expressing, Mutagenesis, Microarray, Genome Wide, Labeling, Generated, Sequencing, Software, Reverse Transcription Polymerase Chain Reaction, Amplification, Plasmid Preparation, Positive Control, Real-time Polymerase Chain Reaction, Over Expression

Journal:

Article Title: Brain Lipid Binding Protein in Axon-Schwann Cell Interactions and Peripheral Nerve Tumorigenesis

doi: 10.1128/MCB.23.6.2213-2224.2003

Figure Lengend Snippet: Mouse neuron-Schwann cell coculture. Anti-BLBP promotes extension of Nf1−/− TXF cell processes along axons. Wild-type (A and B) or Nf1−/− TXF (C and D) mouse Schwann cells labeled with Cell Tracker green were preincubated with rabbit IgG (A and C) or anti-BLBP antibodies (B and D) and seeded onto DRGN cultures stripped of endogenous Schwann cells. Two days after seeding, cocultures were fixed and stained with antineurofilament antibodies followed by Cy3 (red)-conjugated secondary antibodies. Confocal images obtained with Zeiss LSM Image Browser software are shown. Single cells are representative of the majority observed with each treatment. Arrowheads indicate Schwann cell processes. The asterisk indicates the region which is magnified fivefold in the inset. The scale bar in panel C equals 5 μm and also applies to panels A, B, and D. (E) Lower magnification (scale bar, 5 μm) of Nf1−/− TXF on DRGN cultures in the presence of anti-BLBP antibodies. Arrowheads indicate processes from two cells extending along neurites; other cells lack processes. (F) Extension of Nf1−/− TXF cell processes in the presence of anti-BLBP antibodies is statistically significant. The percentages of Nf1−/− TXF cells extending processes along axons in the presence of control IgG (gray bar) or anti-BLBP antibodies (black bar) are graphed. Error bars reflect standard deviations in a Student t test (P = 0.003).

Article Snippet: In DRGN cocultures, neurofilament was visualized by incubation with anti-NF-15g1 antibodies ( 38 ) diluted 1:5, followed by donkey anti-mouse Cy3-labeled secondary antibodies (Jackson Immunoresearch).

Techniques: Labeling, Staining, Software