Introduction 1 2 3 4 4 7 2+ 8 9 10 11 12 13 14 15 16 2+ 17 18 22 18 23 24 7 7 Material and methods Materials 2 4 2 2 Bandeiraea simplicifolia (Griffonia simplicifolia) B. simplicifolia (G. simplicifolia) 1 2 4,5,6 7 3 7 25 1 2 4 12 6 1 Animals Male Wistar rats (280–320 g) were housed under a 12-h light, 12 h-dark cycle and allowed access to lab food and water ad libitum. All procedures using animals were approved by the committee of Animal Care and Use of the relevant local governmental body in accordance with the law of experimental animal protection. Surgery/microinjection 26 1,3 1,11,12,13 4,6 7 7 Immunocytochemical studies and double-immunofluorescence studies 26 27 , Immunocytochemical studies BSI immunoreactivity Free-floating sections were rinsed with 0.05 M Tris-buffered saline (TBS, pH 7.6) and were treated with 1% hydrogen peroxide for 30 min to inactivate endogenous peroxide activity. Immunolabeling was performed with lectin from BSI-B4 (1:200) in TBS containing 2% bovine serum albumin overnight at 4°C, followed by washing in 0.05 M Tris buffer (pH 8.0). Peroxidase activity was visualized with DAB (0.07%) containing nickel ammonium sulphate (1%) and hydrogen peroxide, which renders a black reaction product. Active caspase 3 immunoreactivity Free-floating sections were rinsed with 0.1 M TBS (pH 7.4) and treated with 1% hydrogen peroxide for 25 min to inactivate endogenous peroxide activity. Immunoreactivity (IR) was studied with rabbit anti-active caspase 3 (1:500) in TBS containing 10% normal horse serum (NHS) and 0.1% Triton X-100 overnight at 4°C, followed by biotinylated horse anti-rabbit IgG (1:100, Vector Labs. Burlingame, CA, USA) and preformed streptavidin/biotin-peroxidase complex (1:125, StreptABComplex; DakoCytomation) for 2 h. DAB (0.05%) served as chromogen. GFAP/BrdU immunoreactivity 26 Double-immunofluorescence studies 1 2 4 5 6 7 1 2 4 12 1 6 3 3 7 7 25 7 7 Confocal microscopy Double immunofluorescence was investigated by a scanning confocal microscope (LSM 510, Zeiss, Oberkochen, Germany) equipped with an argon laser emitting at 488 nm (yellow-green Cy2-immunofluorescence) and a helium/neon laser emitting at 543 nm (red Cy3-immunofluorescence). Quantification and statistical analysis 1 Fig. 1 a 1 2 29 b overview c d–f d arrows e, f arrow arrowhead scale bar g p p  1 t Results Immunocytochemistry 1 1 1 1 1 28 1 1 Double-immunofluorescence studies at P2X and P2Y receptor subtypes 1 1,2,4,7 1,2,4,6,12 1,2,4,7 2 1,2 12 4 2 2 4 2 6 2 3,5,6 Fig. 2 1,2,4,7 1,6,12 a–f g–o thin arrow thick arrow scale bars a, b c, d e–i j, k l, m n, o  7 2 29 7 7 25 7 3 3 7 Fig. 3 7 a–d 7 C-term b, d a–d 7 e–j e, f 7 g, h i, j b–g k 1 a p p p  7 7 3 7 3 3 7 3 3 7 7 1 1 Discussion 30 2+ 8 31 32 33 1,3 7 1,2,4,6,12 1–7 1,2,3,4,7 5,6 2,4,6 22 34 35 7 36 1–7 37 37 1 29 2 29 2 4 38 2 4 39 4 7 1 1,2,12 6 4 4 33 4 40 4 4 7 13 16 41 42 7 4 23 43 7 41 44 45 46 7 47 48 1 2,4,6,12 1 49 6 6 50 9 10 51 53 12 12 i/o 10 9 12 12 33 54 12 12 55 1–7 29 1,2,4,6,12 27 1 7 49 1 7 7 56 7 57 7 58 7 59 7 7 7