Journal: Nucleic Acids Research

Article Title: Tyrosine kinase c-Abl couples RNA polymerase II transcription to DNA double-strand breaks

doi: 10.1093/nar/gkz024

Figure Lengend Snippet: Formation and p-Dicer-dependent turnover of dsRNA at DSBs. ( A ) ChIP analysis of GFP-RNaseH1 occupancy at DS1 using site-specific primers. ( B ) Quantitative real-time PCR (qRT-PCT) of DNA immunopurified from DNA–RNA hybrids (DRIP) or upon incubation with recombinant RNaseH at DS1 using S9.6 hybridoma supernatant and region-specific primers. ( C ) qRT-PCR analysis of transcripts associated with CTD Y1P and immunopurification (mNET-IP). Values were normalized to data in absence of GFP-RNaseH1. (A–E) Asterisk, P -value < 0.05, two-tailed t -test. Error bar: mean ± SEM, n = 3. ( D, E ) Imaging and RGB quantitation of CTD Y1P and p-Dicer (p-DCR-1). White box, 2.5× zoom; n , number of cells with shown phenotype in %. ( F ) qRT-PCR of cDNA after J2 immunoselection with (J2 RIP) and reverse transcription with forward (upper panel)- and reverse (lower panel)-oriented primers spanning a region up to 1000 nts distant from DS1 in presence (J2+) or absence (J2-) of 4OHT, or upon preincubation of lysed material with recombinant RNaseIII (J2+ RNaseIII) prior to J2 RIP. J2+ values were set to 1. Asterisk, p-value <0.05, two-tailed t-test. Error bar: mean ±SEM, n = 3. ( G ) Autoradiograph detecting J2 immuno-selected (RNA IP) or total (IN) RNA or pBR322 MspI digest (M) upon end-labeling and PAGE separation. AU, arbitrary units. ( H ) as in (D), but preincubated with Leptomycin B (LMB) and stained with J2. Representative images are shown.

Article Snippet: Cells were permeabilized with PBS/0.3% Tween-20 (10 min, RT), washed 1× in PBS and incubated for 20 min at RT with either BSA (Sigma, 0.2 μg/ml final conc., diluted in PBS containing 0.02 mM NaOAc and 0.2 mM Tris), RNaseA (Sigma, 0.2 μg/ml final conc., diluted in PBS containing 0.02 mM NaOAc and 0.2 mM Tris) or RNaseIII (NEB, 2U final conc., diluted in RNase-free H 2 O containing 1× commercial reaction buffer (NEB) prior to fixation.

Techniques: Real-time Polymerase Chain Reaction, Incubation, Recombinant, Quantitative RT-PCR, Immu-Puri, Two Tailed Test, Imaging, Quantitation Assay, Autoradiography, End Labeling, Staining

Journal: Molecular Brain

Article Title: Human primary mixed brain cultures: preparation, differentiation, characterization and application to neuroscience research

doi: 10.1186/s13041-014-0063-0

Figure Lengend Snippet: Confocal imaging of Human mixed brain culture containing neurons, glia and neural progenitor cells. At each time point, cells were fixed using 4% Paraformaldehyde and dual-labeled with FITC-conjugated Neuronal antibody cocktail (Pan-N, 1:1000) and a Cy3-conjugate of either a glial marker (GFAP, 1:5000) or a neuronal progenitor (Nestin, 1:500). Results at DIV 12, and DIV 32 are shown. Antibodies against Pan-N and or Pan-N and GFAP were used with appropriate secondary antibodies (donkey-anti mouse; 1:500 or Goat-anti rabbit, 1:2000). Confocal fluorescence microscopy was performed on an inverted IX81microscope fitted with a FV1000-MPE and laser launch with confocal excitation with three diode lasers (405, 559 and 635 nm) and an Argon laser (458, 488, 514 nm), controlled by Fluoview v3.0 (Olympus). All imaging was performed with a 60x 1.2 NA water immersion objective. The scale bar is in the bottom right panel, and is applicable to all images.

Article Snippet: Glial Fibrillary Acidic protein (GFAP) (ICC) , 1:10,000 , Sigma , G9269 , Cy3-conj. Donkey anti Rabbit , 1:500 , Jackson Immuno Research , 711-165-152.

Techniques: Imaging, Labeling, Marker, Fluorescence, Microscopy

Journal: Molecular Brain

Article Title: Human primary mixed brain cultures: preparation, differentiation, characterization and application to neuroscience research

doi: 10.1186/s13041-014-0063-0

Figure Lengend Snippet: An increase in neuronal population over time. Human fetal brain (HFB) cells were grown as stated. At each time point, cells were fixed using 4% Paraformaldehyde and dual-labeled with FITC-conjugated Neuronal antibody cocktail (Pan-N, 1:1000) and a Cy3-conjugate of either a glial marker (GFAP, 1:5000) or a neuronal progenitor (Nestin, 1:500). Appropriate secondary antibodies were used. The values at the corners of the boxes refer to percent total of the cells gated for selection that were positive for the appropriate fluorescent-conjugated antibody. LL – Unlabeled cells. UL-Cells positive for Cy3-conjugated marker for either GFAP or Nestin. LR – Cells labeled with Pan-N. UR – Cells positive for either Pan-N and GFAP or Pan-N and Nestin. This data suggests that the stem-ness of the culture reduces over time and the neuronal phenotype beings to emerge.

Article Snippet: Glial Fibrillary Acidic protein (GFAP) (ICC) , 1:10,000 , Sigma , G9269 , Cy3-conj. Donkey anti Rabbit , 1:500 , Jackson Immuno Research , 711-165-152.

Techniques: Labeling, Marker, Selection

Journal: Molecular Brain

Article Title: Human primary mixed brain cultures: preparation, differentiation, characterization and application to neuroscience research

doi: 10.1186/s13041-014-0063-0

Figure Lengend Snippet: List of different primary and secondary antibodies used to characterize the culture

Article Snippet: Glial Fibrillary Acidic protein (GFAP) (ICC) , 1:10,000 , Sigma , G9269 , Cy3-conj. Donkey anti Rabbit , 1:500 , Jackson Immuno Research , 711-165-152.

Techniques: Marker

Journal: Glia

Article Title: Deletion of Tgfβ signal in activated microglia prolongs hypoxia‐induced retinal neovascularization enhancing Igf1 expression and retinal leukostasis

doi: 10.1002/glia.24218

Figure Lengend Snippet: Tgfβ signal deletion in microglia did not affect structure of vessels and vascular development but, promoted the adhesion of leukocytes via enhancing chemoattractant. (a) The schematic shows the protocol that was used to generate data for this figure. (b) GS‐lectin labeled retinal vessels at superficial, intermediate, and deep plexus 8 weeks after tamoxifen injection from P9 to P14. Scale bars = 100 μm. (f) TRITC‐conjugated Concanavalin a (con a) lectin labeled leukocytes adhering to retinal capillary endothelium. Scale bars = 500 μm. (b) the number of branching points per 400 μm 2 was evaluated (control n = 6, Tgfβbr2 KO (ΔMG) n = 7). (c) NG2 positive pericytes at superficial, intermediate, and deep plexus 8 weeks after daily tamoxifen injections on P9 to P14 (scale bars = 100 μm). (e) the number of NG2 positive pericytes per 400 μm 2 was evaluated (control n = 6, Tgfβbr2 KO (ΔMG) n = 5). (f) TRITC‐conjugated Concanavalin a (con a) lectin labeled leukocytes adhering to retinal capillary endothelium (scale bars = 500 μm). (g) Adherent leukocytes to retinal capillaries are visualized by TRITC‐conjugated con a lectin at each layer. There are more leukocytes in all layers in Tgfβbr2 KO (ΔMG) mice compared to control (scale bars = 100 μm). (h) the number of adherent leukocytes labeled with con a lectin in Tgfβbr2 KO (ΔMG) mice was higher than those observed in control mice ( n = 4 each). Data are mean ± SEM. p Values were calculated using a two‐tailed Student's t ‐test, * p < .05.

Article Snippet: The retinal vasculature and adherent leukocytes were imaged by perfusion labeling with TRITC‐conjugated Concanavalin A (Con A) lectin (Vector Laboratories), as described previously (Joussen et al., ; Okunuki et al., ).

Techniques: Labeling, Injection, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Attenuating Diabetic Vascular and Neuronal Defects by Targeting P2rx7

doi: 10.3390/ijms20092101

Figure Lengend Snippet: 3TC protects photoreceptors from age-related degeneration. ( A ) Average retinal thickness across the temporal-nasal axis acquired via quantitative spectral domain optical coherence tomography (SD-OCT), n = 6 eyes per experimental condition. Two-way ANOVA with Tukey’s multiple comparisons test. # p < 0.05, ## p < 0.01, and ### p < 0.001 between non-diabetic untreated and diabetic untreated animals. ( B ) Cryosections from each experimental condition were stained for cone arrestin (red) and counterstained with DAPI (blue). The average number of cones (cone arrestin-positive cells) or rods (DAPI-positive cells in the ONL minus cones) were quantified and normalised to 100 μm of retina length. INL: inner nuclear layer; ONL: outer nuclear layer; OS: outer segment. Scale bar: 25 μm. ( C ) Average number of rods per 100 μm. ( D ) Average number of cones. Data are shown as mean ± SEM. n > 40 images from 3–5 different animals per experimental condition. Two-way ANOVA with Sidak’s test, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Cone arrestin , 1/1000 , Merck Millipore , Cone photoreceptors.

Techniques: Tomography, Staining

Journal: International Journal of Molecular Sciences

Article Title: Attenuating Diabetic Vascular and Neuronal Defects by Targeting P2rx7

doi: 10.3390/ijms20092101

Figure Lengend Snippet: Primary Antibodies and Secondary Antibodies.

Article Snippet: Cone arrestin , 1/1000 , Merck Millipore , Cone photoreceptors.

Techniques: Plasmid Preparation