Smad expression in the in vivo glial scar and in primary astrocytes. A. RNA was isolated from cortex (C), in vivo filter implants (F), untreated (A) and TGF-β1 treated (T) cultured astrocytes. Smad mRNAs were detected by RT-PCR with primers specific for Smads 2, 3 and 4. B. Protein lysates from total brain (B), filter implants (F), cortex (C), untreated (A) and TGF-β1 treated (T) cultured astrocytes were examined for the presence of TGF-β1 activated R-Smads using a Smad 2/3specific antibody. C. Expression of Smads 2 and 3 in filter implants (ii) and cultured astrocytes (iii) was also detected by immunostaining with the same antibody. Similar immunostaining results were obtained with a specific antibody recognizing Smad2 in filter implants (iv) and in primary astrocyte cultures (v). GFAP immunohistochemistry (i) was performed to demonstrate reactive astrogliosis elicited by filter implantation. Scale bars in (i), (ii) and (iv) are 75 μm. Magnification for (iii) is 500× and for (v) is 200×.