Introduction 1 3 4 5 5 4 6 5 7 9 10 11 12 13 14 15 16 17 18 19 20 12 21 23 Materials and methods Materials N N D N 6 N p 16 17 1 Animals cd39 24 Müller cell swelling 25 2 2 Calcium imaging 2 Immunohistochemistry Isolated rat retinas were fixed in 4% paraformaldehyde for 2 h. After several washing steps in buffered saline, the tissues were embedded in saline containing 3% agarose (w/v), and 60-μm-thick slices were cut by using a vibratome. For double labelling, the slices were incubated in 5% normal goat or donkey serum plus 0.3% Triton X-100 in saline for 2 h at room temperature and, subsequently in a mixture of primary antibodies overnight at 4°C. After washing in 1% bovine serum albumin, secondary antibodies were administered for 2 h at room temperature. Control slices were stained without primary antibodies; no unspecific labelling was observed following incubation with secondary antibodies (not shown). Images were taken with the LSM. Immunocytochemistry To prepare suspensions of dissociated cells, isolated rat retinas were stored for 30 min in saline containing 0.4 mg/ml papain (Boehringer, Mannheim, Germany) at 37°C. After fixation with 4% paraformaldehyde for 10 min and washing with saline, the tissues were triturated with a pipette until single cells were dissociated. The cells were blocked and permeabilised for 30 min with 5% normal goat serum and 0.3% Triton-X 100, followed by incubation with primary antibodies for 6 h at 4°C. After washing in 1% bovine serum albumin in saline, the secondary antibodies were applied for 1 h at room temperature. Enzyme histochemistry 16 3 2 2 2 4 2 Data analysis F F 0 F 0 t Results Purinergic signalling inhibits osmotic swelling of Müller glial cells 26 27 1 6 1 1 1 1 1 1 1 1 1 Fig. 1 c–h a Insets left right Bar b c TTX d e 1 f g N N D h N Bar diagrams n P P •• P ••• P ○○ P ○○○ P  1 28 29 30 1 1 1 ATP-evoked calcium responses 31 1 31 6 32 2 33 34 2 Fig. 2 a Asterisk Arrow Arrowhead Bar b N N D c n P  Immunolocalisation of ectonucleotidases 3 3 3 3 3 3 3 3 Fig. 3 a b c Filled arrows Filled arrowheads INL Unfilled arrows Unfilled arrowheads a, c ONL b GCL IPL PRS Bars  1 3 1 3 1 26 6 3 Ectonucleotidase histochemistry 4 3 4 3 4 4 35 4 Fig. 4 cd39 cd39 a b arrows c p GCL INL IPL ONL PRS Bars  Discussion 6 6 1 1 1 1 31 36 1 1 1 6 37 1 31 32 4 7 9 16 17 19 38 14 15 39 1 35 40 42 43 35 44 5 5 5 32