The Journal of Comparative Neurology SINGLE CELL TRANSCRIPTIONAL PROFILE OF MGCS In situ hybridization, Xgal histochemistry, and immunohistochemistry RNA tissue hybridization was performed following the detailed protocol described (Trimarchi et al., 2007), which is based on previous techniques developed by Murtaugh et al. (1999). Retinae were dissected from young adult ( P21) C57/BL6 or CD1 mice. Tissues were ﬁxed in 4% paraformaldehyde (PFA) in PBS (pH 7.4), dehydrated, and embedded in parafﬁn, or cryoprotected in 30% sucrose in PBS and embedded in Tissue-Tek OCT compound. In situ hybridization (ISH) was performed on 20- m retinal sections. The protocol used for dissociated cell in situ hybridization (DISH) was performed as in Blackshaw et al. (2004) and Trimarchi et al. (2007), in combination with immunohistochemistry. For antibody incubation and de- tection, slides were incubated in blocking buffer (PBS, 0.1% Triton-X100, and 5% goat serum) for 30 minutes at room temperature in a humidiﬁed chamber. Primary an- tibody (anti-Glul, mouse monoclonal antibody against glu- tamine synthetase; Chemicon/Millipore, Temecula, CA, MAB302; as immunogen glutamine synthetase puriﬁed from sheep brain) was added 1:500 diluted in PBS and incubated at room temperature for 1.5 hours. This anti- body recognizes a single 45-kDa protein in adult retinal tissue by Western blot analysis (Chang et al., 2007), and staining of adult murine retinal sections revealed a char- acteristic glial expression pattern (Rhee et al., 2007). Slides were then washed three times with PBT (PBS and 0.1% Tween-20), and then cy2-labeled secondary an- tibody (donkey anti-mouse IgG, cyanine cy2, Jackson Im- munoResearch, West Grove, PA, 715-225-150) in PBT (1: 250) was added. The slides were incubated at room temperature for 1 hour and afterwards washed again three times with PBT. At the end, slides were DAPI- stained and mounted in gel/mount (Biomeda, Foster City, CA). For immunohistochemistry on 20- m OCT retinal sections, slides were blocked in blocking buffer (PBS, 0.1% Triton-X100, and 10% goat serum) for 45 minutes at room temperature in a humidiﬁed chamber. Primary antibody, polyclonal anti-Pax6 (puriﬁed rabbit antibody; Covance, PRB-278P; generated against the peptide QVPGSEP- DMSQYWPRLQ of the mouse Pax6 protein and puriﬁed on a peptide afﬁnity column; Marquardt et al., 2001), and monoclonal anti-Glul (Chemicon/Millipore, MAB302) were added at 1:150 and 1:500, respectively, diluted in blocking buffer, and incubated overnight at 4°C. The anti-Pax6 antibody reacts strongly with a single band of approxi- mately 50 kDa on Western blots of retinal tissue extracts (Davis and Reed, 1996). Slides were then washed three times with PBT (PBS and 0.1% Triton-X100), and then cy2-labeled anti-mouse secondary antibody (Jackson ImmunoResearch, 715-225- 150) and cy3-labeled anti-rabbit secondary antibody (don- key anti-rabbit IgG, cyanine cy3, Jackson ImmunoRe- search, 711-165-152) in blocking buffer (1:250) were added. The slides were incubated at room temperature for 3 hours and afterwards washed again three times with PBT. Slides were DAPI-stained and mounted in gel/ mount. For Xgal staining, retinal tissue was harvested and ﬁxed with a 1% PFA/0.5% glutaraldehyde mix (Kwan et al., 2001). Retinae were stained as whole mounts (Turner et al., 1990) and sectioned afterwards. 227 Photography Photography of immunohistochemical staining patterns on sections was done on an upright confocal microscope (Leica TCS SP2). Pictures of ISH and DISH experiments were acquired on either a Zeiss Axiophot microscope or a Nikon eclipse E1000 microscope, both equipped with a Ni- kon DXM 1200F digital camera. Brightness and contrast of the section ISH and DISH images were adjusted by using Adobe Photoshop 7.0 (San Jose, CA). Digital magniﬁcation was also done by using Adobe Photoshop 7.0. Generation of Pdgfra BAC transgenic mice The Cre recombinase gene, followed by the SV40 late polyA site and an FRT-ﬂanked kanamycin cassette, was cloned into a pBSKS vector (Cre-pA-kan). Cre-pA-kan was ampliﬁed by PCR primers containing the following 50-bp homology arms and NotI sites (Lee et al., 2001; Liu et al., 2003): CGCCCCGCGGCCGCGGCTAATGCT- GTTTATGTTTGGCTTTTGTCATTTGCAGGTCTCAGG- AGCTATGCCCAAGAAGAAGAGGAAGGTGTCCAA and ATAATTGCGGCCGCATACCTGTGAGGAGACAGCTGA- GGACCAGAAAGACCTGGTGGGAGGTCCCTCCCGGC- GGATTTGTCCTACTCAGGAGAGCG. The PCR product was digested by NotI and cloned into the NotI sites of pBSKS (pdg-Cre-pA-kan-pdg). The pdg-Cre-pA-kan-pdg was digested by NotI and recombined into the RP23- 116F7 BAC clone; the loxP sites of the recombined BAC were removed, and the BAC was linearized with AscI before pronuclear injection (Lee et al., 2001; Liu et al., 2003). The BAC was injected into C57/BL6 blastocysts, and transgenic animals were afterwards crossed to C57/ BL6 animals. Animals CD1 and C57/BL6 mice were purchased from Charles River Laboratories (Wilmington, MA). The JAX® transgenic strain B6.129P2-Gpr37tm1Dgen/J was purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were genotyped for the targeted allele by using DNA extracted from tail preparations, and PCR ampliﬁcation with the following primer pairs was performed. For the targeted allele, the gene-speciﬁc 5 primer, GGCCAAGAGAGAATTGGAGAT- GCTC, and the 3 primer in neo, GGTGGATTAGAGCTTA- AATGCCTGCTCT, were used. For the endogenous allele, the gene-speciﬁc 5 primer, GGCCAAGAGAGAATTGGA- GATGCTC, and the gene-speciﬁc 3 primer, AACGGGTCT- GCAGATGACTGGGTTC, were used. Mice carrying the Pdgfra-CRE BAC transgene or the RC::PFwe allele (gift of A. Farago and S. Dymecki; Farago et al., 2006) were crossed to generate mice hemizygous for PDGFRa-CRE and hemizygous for the RC::PFwe allele. RESULTS Gene expression proﬁle of single M u¨ller glial cells Adult murine retinae were dissociated to single cells by using papain digestion. Within less than 2 hours of dissection, individual M u¨ller glial cells were identiﬁed by their distinctive morphology (Fig. 1) and picked from a dish of dissociated cells with a micropipette. They were then washed, lysed, and subjected to reverse tran- scription. After a terminal transferase reaction to add A’s to the 3 end of the cDNA, 35 rounds of PCR were