Journal: eLife

Article Title: Mesenchymal stromal cell aging impairs the self-organizing capacity of lung alveolar epithelial stem cells

doi: 10.7554/eLife.68049

Figure Lengend Snippet:

Article Snippet: Other , Mouse genotyping service , Transnetyx, Inc, Cordova, TN , , .

Techniques: Staining, Isolation, Recombinant, Plasmid Preparation, Protein Quantitation, Knock-Out, Inverted Microscopy, Microscopy, Cell Analysis, Purification, Software, Sequencing

Journal: Journal of Neurotrauma

Article Title: Kir6.2, the Pore-Forming Subunit of ATP-Sensitive K + Channels, Is Overexpressed in Human Posttraumatic Brain Contusions

doi: 10.1089/neu.2017.5619

Figure Lengend Snippet: Antibodies Used for Immunohistochemistry

Article Snippet: 9 , 33 As shown in , the cardiomyocytes were strongly positive for Kir6.2. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 1. caption a7 Antibody validation. (A) Upper images show the result of a western blot analysis of human Kir6.2 in five specimens of human brain tissue (lanes 1 to 5: 2, 4 corresponding to contusion specimens and 1, 3, 5 corresponding to healthy tissue) using two independent antibodies (Alomone Ab and GeneTex Ab).

Techniques:

Journal: Journal of Neurotrauma

Article Title: Kir6.2, the Pore-Forming Subunit of ATP-Sensitive K + Channels, Is Overexpressed in Human Posttraumatic Brain Contusions

doi: 10.1089/neu.2017.5619

Figure Lengend Snippet: Antibody validation. (A) Upper images show the result of a western blot analysis of human Kir6.2 in five specimens of human brain tissue (lanes 1 to 5: 2, 4 corresponding to contusion specimens and 1, 3, 5 corresponding to healthy tissue) using two independent antibodies (Alomone Ab and GeneTex Ab). The bar graphs are shown as a visual aid for the inmunoblot results. Optical density with both antibodies was compared and resulted in a Pearson correlation coefficient of 0.88 (p = 0.049). The scatterplot is shown as a visual aid to the immunoblot results. (B) Fluorescence labeling of Kir6.2 using Alomone Ab (red) and SantaCruz Ab (green); merged images in yellow. (C) Western blot analysis of a human recombinant Kir6.2 protein (ab114436; Abcam, Cambridge, UK) (1) tagged with green fluorescent protein (GFP) and GFP recombinant protein (2) used as a negative control. (D) Kir6.2 expression (green) in a cell line of human cardiomyocytes (P10451; Innoprot, Derio, Spain) used as a positive control. Nuclei were counterstained with 4,6-diamino-2-phenylindole (DAPI).

Article Snippet: 9 , 33 As shown in , the cardiomyocytes were strongly positive for Kir6.2. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 1. caption a7 Antibody validation. (A) Upper images show the result of a western blot analysis of human Kir6.2 in five specimens of human brain tissue (lanes 1 to 5: 2, 4 corresponding to contusion specimens and 1, 3, 5 corresponding to healthy tissue) using two independent antibodies (Alomone Ab and GeneTex Ab).

Techniques: Western Blot, Fluorescence, Labeling, Recombinant, Negative Control, Expressing, Positive Control

Journal: Journal of Neurotrauma

Article Title: Kir6.2, the Pore-Forming Subunit of ATP-Sensitive K + Channels, Is Overexpressed in Human Posttraumatic Brain Contusions

doi: 10.1089/neu.2017.5619

Figure Lengend Snippet: Cross-reactivity Kir6.2-Kir6.1. (A) Staining of Kir6.1 (green; APC-105; Alomone Labs) and Kir6.2 (red; APC-020; Alomone Labs) in the same section of mouse brain tissue. Kir6.1 shows labeling in blood vessel-like structures and Kir6.2 in neuronal-like structures. This shows that the Kir6.2 antibody does not cross-react with the Kir6.1 epitope. (B) Western blot analysis of a lysate of HEK293T-cells transfected with an empty vector (lane 1) and HEK293T cells that overexpress Kir6.1 (NBL1-12174; Novus Biological, Littleton, CO) (lane 2). The membrane shown on the left was incubated with an anti-Kir6.1 antibody and the one on the right with an anti-Kir6.2 antibody. Only the Kir6.1 antibody was detecting the HEK293T-cells lysate showing that cross-reactivity does not occur between the Kir6.2 antibody and the Kir6.1 epitope.

Article Snippet: 9 , 33 As shown in , the cardiomyocytes were strongly positive for Kir6.2. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 1. caption a7 Antibody validation. (A) Upper images show the result of a western blot analysis of human Kir6.2 in five specimens of human brain tissue (lanes 1 to 5: 2, 4 corresponding to contusion specimens and 1, 3, 5 corresponding to healthy tissue) using two independent antibodies (Alomone Ab and GeneTex Ab).

Techniques: Staining, Labeling, Western Blot, Transfection, Plasmid Preparation, Incubation

Journal: Journal of Neurotrauma

Article Title: Kir6.2, the Pore-Forming Subunit of ATP-Sensitive K + Channels, Is Overexpressed in Human Posttraumatic Brain Contusions

doi: 10.1089/neu.2017.5619

Figure Lengend Snippet: Kir6.2 protein levels in control and contusion samples. (A) Box plots of Kir6.2 relative intensity in control and contusion samples, showing a significant increase of Kir6.2 expression (p = 0.014) in contusion specimens; data normalized to β-actin loading controls. (B) Representative image of a western blot showing a clear difference between control and contusion samples.

Article Snippet: 9 , 33 As shown in , the cardiomyocytes were strongly positive for Kir6.2. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 1. caption a7 Antibody validation. (A) Upper images show the result of a western blot analysis of human Kir6.2 in five specimens of human brain tissue (lanes 1 to 5: 2, 4 corresponding to contusion specimens and 1, 3, 5 corresponding to healthy tissue) using two independent antibodies (Alomone Ab and GeneTex Ab).

Techniques: Expressing, Western Blot

Journal: Journal of Neurotrauma

Article Title: Kir6.2, the Pore-Forming Subunit of ATP-Sensitive K + Channels, Is Overexpressed in Human Posttraumatic Brain Contusions

doi: 10.1089/neu.2017.5619

Figure Lengend Snippet: Expression of Kir6.2 in different cell types. The first three columns of the montage show Kir6.2 expression in different cell types of the peri-contusional tissue and the last three show Kir6.2 expression in control tissue. Peri-contusional tissue: fluorescent double labeling for NeuN/ glial fibrillary acidic protein (GFAP)/Iba1/CD31 (green) and Kir6.2 (red). Merged images are presented in the third column. Control tissue: fluorescent double labeling for NeuN/GFAP/Iba1/CD31 (green) and Kir6.2 (red). Merged images are presented in the last column. In controls, the Kir6.2 expression was evident in neurons and microglia but was minimal in astrocytes (GFAP-positive cells). Original magnification = 100x. Scale: 20 μm. Nuclei were counterstained with 4,6-diamino-2-phenylindole (DAPI).

Article Snippet: 9 , 33 As shown in , the cardiomyocytes were strongly positive for Kir6.2. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 1. caption a7 Antibody validation. (A) Upper images show the result of a western blot analysis of human Kir6.2 in five specimens of human brain tissue (lanes 1 to 5: 2, 4 corresponding to contusion specimens and 1, 3, 5 corresponding to healthy tissue) using two independent antibodies (Alomone Ab and GeneTex Ab).

Techniques: Expressing, Labeling

Journal: Journal of Neurotrauma

Article Title: Kir6.2, the Pore-Forming Subunit of ATP-Sensitive K + Channels, Is Overexpressed in Human Posttraumatic Brain Contusions

doi: 10.1089/neu.2017.5619

Figure Lengend Snippet: Kir6.2 expression in neurons, glial cells, and microglia. From left to right, representative box plots of Kir6.2 expression in neurons (percentage), in glial cells, and microglia (semi-quantitative scale from 0 to 3). The results show a significantly increased expression of Kir6.2 in glial cells from contusion samples (Wilcoxon rank sum test; p = 0.03), but non-significant differences in neurons and microglia. Asterisks indicate statistically significant differences (*p ≤ 0.05).

Article Snippet: 9 , 33 As shown in , the cardiomyocytes were strongly positive for Kir6.2. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 1. caption a7 Antibody validation. (A) Upper images show the result of a western blot analysis of human Kir6.2 in five specimens of human brain tissue (lanes 1 to 5: 2, 4 corresponding to contusion specimens and 1, 3, 5 corresponding to healthy tissue) using two independent antibodies (Alomone Ab and GeneTex Ab).

Techniques: Expressing

Journal: Mobile DNA

Article Title: Detecting variable (V), diversity (D) and joining (J) gene segment recombination using a two-colour fluorescence system

doi: 10.1186/1759-8753-1-9

Figure Lengend Snippet: Physiological variable (V), diversity (D) and joining (J) recombination and analogous recombination substrates . (a) V and J segments on opposite strands (as found in the human Igκ locus) are joined by inversion between the recombination signal sequence (RSS)12 (filled triangle) and RSS23 (open triangle) motifs to generate a linked signal joint and coding joint (the VJ rearrangement). (b) V and J segments located on the same strand (as found in the human Igκ and λ loci) are recombined by deletion of the intervening DNA, leaving the coding segment on the chromosome and the signal joint on an excised circle of DNA. (c) In the inversion substrate, the DsRed gene and the EGFP gene are located on opposite stands, flanked by RSS12 and RSS23 motifs. V(D)J recombinase activity flips the segment allowing DsRed to be replaced by EGFP. (d) In the deletion substrate, the RSS motifs are in opposite orientations and flank the DsRed gene. On recombination, the DsRed gene is deleted, placing EGFP adjacent to the promoter. A single promoter is present in both constructs (curved arrow). The positions of the primer sequences F1 and R1, which were used to analyse recombination at the DNA level and for RT-PCR analysis, are shown.

Article Snippet: Cells transfected with deletion or inversion substrates were analysed for the expression of the EGFP protein (resulting from recombination of the substrate) using the FL1 channel of a FACS Calibur flow cytometer (BD Biosciences, Oxford, UK).

Techniques: Sequencing, Activity Assay, Construct, Reverse Transcription Polymerase Chain Reaction

Journal: Mobile DNA

Article Title: Detecting variable (V), diversity (D) and joining (J) gene segment recombination using a two-colour fluorescence system

doi: 10.1186/1759-8753-1-9

Figure Lengend Snippet: Cell-type specificity of recombination . (a) Recombination activating gene (RAG)1 and RAG2 expression in pre-B 300-19P cells (REC+) and WEHI231 B cells (REC-) analysed by reverse transcription polymerase chain reaction (RT-PCR). Actin transcripts were detected in both cell lines. The RAG genes are intronless and assays were performed using reverse transcriptase (RT+) as well as in its absence (RT-) to ensure that signals were not derived from contaminating genomic DNA. (b) Detection of recombination by PCR (using the F1 and R1 primers shown in Figure 1). A PCR product of 155 bp was predicted following recombination. (c) RT-PCR to detect expression of EGFP mRNA from the transfected substrates. The PCR product diagnostic of EGFP transcripts from the recombined substrate is 155 bp. The 975-bp product detected from the deletion substrate transcripts represents a mRNA that traverses the entire DsRed gene and continues on through the EGFP gene. No products were detected in the absence of reverse transcriptase (- RT enzyme). Both DNA recombination and mRNA expression assays were performed from untransfected cells (Untrans) and in cells stably transfected with the deletion and inversion substrates (non-clonal populations grown under continuous selection) using 300-19P (REC+) and WEHI231 (REC-) cells. A (-) symbol indicates no added template.

Article Snippet: Cells transfected with deletion or inversion substrates were analysed for the expression of the EGFP protein (resulting from recombination of the substrate) using the FL1 channel of a FACS Calibur flow cytometer (BD Biosciences, Oxford, UK).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Transfection, Diagnostic Assay, Stable Transfection, Selection

Journal: Mobile DNA

Article Title: Detecting variable (V), diversity (D) and joining (J) gene segment recombination using a two-colour fluorescence system

doi: 10.1186/1759-8753-1-9

Figure Lengend Snippet: Expression of the recombined substrates . (a) Fluorescence microscopy of EGFP and DsRed expression. The four panels show expression of the DsRed (from unrearranged template) and rearranged EGFP gene products from inversion and deletion substrates in both 300-19P and WEHI231 cells. (b) Flow cytometric analysis of 4-week cultures of untransfected and transfected recombinant 300-19P (REC+) and WEHI231 (REC-) cell lines. The fold increase in number of EGFP-expressing cells was established by assigning an arbitrary value of 1 for untransfected cells.

Article Snippet: Cells transfected with deletion or inversion substrates were analysed for the expression of the EGFP protein (resulting from recombination of the substrate) using the FL1 channel of a FACS Calibur flow cytometer (BD Biosciences, Oxford, UK).

Techniques: Expressing, Fluorescence, Microscopy, Transfection, Recombinant