In the first study 5 of our antibodies are used. It investigates the expression of purinergic P2 receptors in oxygen-induced retinal neovascularization.
Sylvia Sarman; Jorge Mancini; Ingeborg van der Ploeg; J. Oscar Croxatto; Anders Kvanta; Juan E. Gallo. Involvement of Purinergic P2 Receptors in Experimental Retinal Neovascularization. Current Eye Research. 10.1080/02713680701885470 .
...Five eyes (5 animals) from either hyperoxa- or non-hyperoxia-treated mice were enucleated and fixed for 48 hr at 4°C in paraformaldehyde (Sigma-Aldrich, St Louis, MO, USA). Afterward, eyes were immersed for cryoprotection in graded sucrose solution (5% overnight, 10%, 15%, and 20%) and interlocked with resin. Fourteen micron sections were obtained (Shandon AS325 Retraction Microtome, Thermo Scientific, Waltman, MA, USA) and fixed on polylysine-treated glass slides. After overnight incubation in primary antibody (P2X1 1:1000, P2X2 1:1000,P2X3 1:1000, and P2Y2 1:800) (Neuromics, Minneapolis, MN, USA) at 4°C, sections were treated with biotinylated secondary antibodies followed by an avidin peroxidase complex step (Vectastin Elite ABC, Vector Laboratories, Burlingame, CA, USA). Finally, a color reaction was obtained using 3,3'-diaminobenzidine (DAB)/nickel-enhanced solution for staining (Sigma-Aldrich, St Louis, MO, USA). At least 6 sections per retina were analyzed. For immunofluorescence, the sections were labeled with lissamine rhodamine-conjugated goat anti-rabbit Ig-G or with fluorescein-5-isothiocyanate-conjugated goat anti-guinea pig Ig-G (Jackson Immunoresearch Laboratories, West Grove, PA, USA). At least 6 sections per retina were analyzed. Visualization was done using a Nikon Fluorescence Eclipse Microscope (Tokyo, Japan), and photographs were taken with a Nikon DN 100 Digital Camera (Tokyo, Japan)...
Images: Expression of P2X and P2Y receptors in normal mice and in a model of ischemia-
induced pathological retinal angiogenesis as assessed by immunohistochemistry. (A) P2X2 receptor expression was found in the outer plexiform layer of control mice. (B) In micetreated with oxygen, the expression was similarly found in the outer plexiform layer. In addition, a strong signal was also found in the inner plexiform layer. This induction was noted in all slides analyzed. (C) Retina without primary antibody for P2X2. (D) In control mice,
weak P2Y2 receptor expression was seen in the ganglion cell and in the nerve fiber layer. (E) In mice treated with oxygen, P2Y2 expression
showed a similar overall distribution as control mice. The signal was, however, strongly up-regulated. This increase was noted in all slides analyzed. (F) Retina without primary antibody for P2Y2. Black bar = 20 μm.
The second studies P2Xs' role in neurotransmision.
Elsa Fabbretti*, Elena Sokolova*, Lara Masten, Marianna D’Arco, Alessandra Fabbro, Andrea Nistri and Rashid Giniatullin. Identification of negative residues in the P2X3 ATP receptor ectodomain as structural determinants for desensitization and the Ca2+ sensing modulatory sites. JBC Papers in Press. Published on October 8, 2004 as Manuscript M409772200.
...Western immunoblots of transfected or untransfected HEK cells were performed as recently reported (19). In brief, these were lysed using a buffer containing 100 mM Tris HCl (pH 6.8), 200 mM dithiothreitol, 4 % SDS, 20 % glycerol and a cocktail of protease inhibitors (Sigma), and separated on 10 % polyacrylamide gel. After blocking with Tris saline buffer (TBS) containing milk, Tween 20 and preimmune serum, proteins were incubated overnight at 4°C with an anti-P2X3 antibody (Neuromics; 1:2000). Immunocomplexes were incubated for 1 h with a peroxidase-conjugated secondary antibody (Sigma; 1:4000) and detected with a chemiluminescence ECL kit (Amersham). Negative controls were obtained by mock transfection of HEK cells with the pEGFP-N1 plasmid (CLONTECH). Controls for efficient loading of different lysate materials were carried out by using an anti-β actin antibody (mouse monoclonal, 1:2000, Sigma)...
Related Reagents:
P2X1-Rabbit Antibody
P2X2-Guinea Pig Antibody
P2X2-Rabbit Antibody
P2X3-Rabbit Antibody
P2X3-Guinea Pig Antibody
Related Reagents:
All Purinergic Receptor Antibodies
All Pain and Inflammation Antibodies
Vision and Retina Research Antibodies
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