fpr3 sense and antisense probes  (Biotechnology Information)

Journal: The Journal of Biological Chemistry

Article Title: Strain-specific Loss of Formyl Peptide Receptor 3 in the Murine Vomeronasal and Immune Systems *

doi: 10.1074/jbc.M116.714493

Figure Lengend Snippet: Fpr3 is expressed in vomeronasal sensory neurons and immune cells. A, left, in situ hybridization with Fpr3 sense and antisense probes on coronal slices of the vomeronasal organ. Black triangles mark Fpr3-positive cells. Scale bar, 100 μm. Right, RT-PCR analysis of Fpr3 expression in the vomeronasal organ. A band of the correct size and sequence was observed in VNO cDNA (+RT) but not in the negative control without reverse transcriptase (−RT). Similar results were obtained in five independent experiments. Size marker (L) FastRuler Middle Range DNA Ladder. B, left, representative immunostaining with Fpr3-ECL2 on dissociated vomeronasal cells of C57Bl/6NCrl mice. The white triangle marks a Fpr3-positive cell (red). Right, quantification of Fpr3 immunoreactivity. Average frequency of Fpr3 expression was analyzed in a total of 60,426 cells from six independent experiments. Antibody specificity was demonstrated by blocking the specific binding site through preincubation with 10 μg/ml of the peptide used for antibody generation. C, colocalization of Fpr3 (red) with different cellular markers (green) on dissociated vomeronasal cells from C57Bl/6NCrl mice using phosphodiesterase 4A (PDE4A) as a marker for the apical zone of the VNO, vomeronasal type 2 receptor 2 (V2R2) as a marker for Vmn2r-expressing cells, G protein subunit Gαo as a marker for the basal zone, and OMP as a marker for mature VSNs. D, colocalization of Fpr3 (red) with immune cell markers (green) on dissociated cells. Fpr3 staining colocalized with Ly6G (lymphocyte antigen 6G), a neutrophil marker. No colocalization was seen for CD45R (cluster of differentiation molecule 45R) that is expressed by most immune cells but absent in neutrophils. In all colocalization experiments, the colocalizing cells are marked with a white triangle and non-colocalizing with a white arrow. All cells were counterstained with the nuclear dye Hoechst33342 (blue). Insets show Fpr3-positive cells in a ×3 magnification. Scale bars, 20 μm. The bar chart below each picture denotes the quantification from two to five experiments, analyzing the colocalizations in a total of 7000 to 57,000 cells. Precise numbers are given in the graphs. Fpr3-positive cells that coexpress the marker are labeled by +, and cells not coexpressing the marker are labeled with −. Values in parentheses denote positive versus total cells. Error bars, S.D.

Article Snippet: In Situ Hybridization Fpr3 sense and antisense probes (nucleotide 672–1056) from National Center for Biotechnology Information database, accession number NM_008042.2 plus 153 bp of the 3′-untranslated region were synthesized from a full-length Fpr3 DNA template using a digoxigenin-labeling procedure during in vitro transcription.

Techniques: In Situ Hybridization, Reverse Transcription Polymerase Chain Reaction, Expressing, Sequencing, Negative Control, Reverse Transcription, Marker, Immunostaining, Blocking Assay, Binding Assay, Staining, Labeling