To examine glutamine synthetase expression in relation to GFAP, vimentin, and αSMA, freshly isolated porcine Müller cells were maintained in culture for varying lengths of time and then probed with the use of indirect immunofluorescence. As we reported previously, Müller cells in retina and all tissue culture stages were strongly positive for vimentin (not shown).
15 16 Normal retina probed with antibodies against GFAP revealed astrocytes and radially oriented Müller cells
(Fig. 1A) , whereas glutamine synthetase labeling was limited to Müller cells alone (
Fig. 1B , same field as 1A). In contrast, the adjacent section probed for GFAP and αSMA was similarly positive for GFAP
(Fig. 1C)but negative for αSMA except for vascular myocytes (
Fig. 1D , same field as 1C). Freshly isolated Müller cells maintained in culture for 1 day had similar patterns of immunoreactivity in that all cells were positive for GFAP and glutamine synthetase (
Figs. 1E and 1F , same fields) and negative for αSMA (1H, same field as 1G). Cells maintained in culture for 7 days were of an intermediate phenotype in that the cells were less strongly positive for GFAP and had little or no glutamine synthetase-associated signal (
Figs. 1I and 1J , same field). In contrast, the cells were now variably positive for αSMA and, in some cases, had prominent stress fibers (
Figs. 1K and 1L , same field). Finally, as previously reported, Müller cells at 35 days in culture (passage 5) were uniformly negative for GFAP and positive for αSMA (not shown).
15 16 These cells were also negative when probed for the presence of glutamine synthetase (not shown).