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Supporting Information Supporting Information Biagioli et al. 10.1073/pnas.0813216106 SI Text and the procedure was repeated twice. Single cells were then RNA Isolation, Reverse Transcription, qPCR, Cloning, and Sequencing. resuspended in panning buffer and incubated on lectin-coated RNA was extracted from cell lines and blood by using TRIzol panning plates for 15 min at room temperature. Nonadherent reagent (Invitrogen) and following vendor instructions. cells were transferred to the next panning plate (four pannings, RNA was extracted from LCM- or FACS-purified cells with an 15 min each). Then, nonadherent cells were collected, centri- Absolutely RNA Nanoprep Kit (Stratagene). Single-strand fuged, and resuspended in serum-free neuronal medium or PBS. cDNA was obtained from purified RNA by using the iSCRIPT A similar procedure was also followed for the dissociation of cDNA Synhesis Kit (Bio-Rad) according to the manufacturer’s cortical and hippocampal astrocytes and oligodendrocytes. instructions. Quantitative RT-PCR was performed by using A cell strainer with 70-␮m nylon mesh was used to obtain a SYBER-Green PCR Master Mix and an iQ5 Real-Time PCR single-cell suspension (BD Falcon) before sorting. 7-AAD Detection System (Bio-Rad). (Beckman–Coulter) was added to the cell suspension to exclude Quantitative RT-PCR was performed with an iCycler IQ dead cells. A high-speed cell sorter (MoFlo) was used to sort (Bio-Rad); ␤-actin was used as an endogenous control to nor- subpopulation of cells expressing GFP. Sorting parameters used malize the expression level of target genes. Primers were chosen for the three different populations are visualized in Fig. S8. by using the software Beacon Designer 2.0 (Biosoft Interna- tional). Primer sequences are available on request. Results were Immunocytofluorescence and Immunohistofluorescence. For immu- normalized to ␤-actin, and the initial amount of the template of nofluorescence experiments cells were fixed in 4% paraformal- each sample was determined as relative expression versus one of dehyde (Tousimis Research) directly added to culture medium the samples chosen as reference. The relative expression of each for 10 min, then washed with PBS two times, treated with 0.1 M sample was calculated by the formula 2expϪ⌬⌬Ct (User Bulletin glycine for 4 min in PBS, and permeabilized with 0.1% Triton 2 of the ABI Prism 7700 Sequence Detection System). X-100 in PBS for another 4 min. After washing with PBS and PCR and qPCR amplicons were cloned in pGEM-TEasy blocking with 0.2% BSA, 1% NGS, and 0.1% Triton X-100 in vector (Promega) and sequenced with T7/Sp6 primers with a PBS (blocking solution), cells were incubated with the indicated LI-COR Global Edition DNA sequencer. antibodies diluted in blocking solution for 90 min at room temperature. After washes in PBS, cells were incubated with In Situ Hybridization. After perfusion with 4% paraformaldehyde labeled secondary antibodies for 60 min. For nuclear staining, in PBS, the mouse brain was removed, postfixed, and cryopro- cells were incubated with 1 ␮g/mL DAPI for 5 min. Cells were tected overnight at 4 °C in 30% sucrose. In situ hybridization was washed and mounted with Vectashield mounting medium (Vec- performed on criostat slices (16 ␮m). tor). Sense and antisense probes were generated by in vitro tran- For immunohistochemistry, 8- to 12-week-old C57/B6 mice scription from the cDNA encoding mouse ␤-globin (Jackson Laboratories) were deeply anesthetized and intensively (NM࿝008220.3, gene nucleotides ϩ27 to ϩ596; 569 bp: complete perfused transcardially with PBS followed by 4% paraformal- CDS, 28 bp in the 5Ј UTR, 97 bp in the 3Ј UTR), and mouse dehyde diluted in PBS. Brains were removed and postfixed in 4% ␣-globin (NM࿝008218.2, gene nucleotides ϩ12 to ϩ521; 509 bp: paraformaldehyde for1hatroom temperature. The region complete CDS, 24 bp in the 5Ј UTR, 47 bp in the 3Ј UTR). containing the SN was isolated and cut with a vibratome (40 Probes were labeled with digoxigenin (DIG) by using DIG RNA ␮m). Slides were blocked with PBS, 10% NGS, 1% BSA, and 1% Labeling Mix (Roche Applied Science), and the digoxigenin fish gelatin (filtered) for1hatroom temperature, and the incorporation was tested by Northern blot analysis. Hybridiza- primary and secondary antibodies were diluted in PBS, 1% BSA, tion was performed with probes at a concentration of 4 ␮g/mL 0.1% fish gelatin, and 0.3% Triton X-100. Incubation with at 60 °C for 16 h. Anti-DIG AP conjugate (Roche Applied primary antibodies was performed for 16 h at room temperature; Science) was used as the detection antibody, and colorimetric incubation with secondary antibodies was performed for2hat reactions were performed with NBT/BCIP solution (Sigma– room temperature. Nuclei were labeled with DAPI. Slides were Aldrich). Sections were then washed, incubated with primary mounted with mounting medium for fluorescence Vectashield antibody for3hatroom temperature and with secondary (Vector Lab) and observed with a confocal microscope. antibody for1hatroom temperature. Sections were mounted Immunohistofluorescence on human postmortem brain sec- with Vectashield and observed at fluorescence microscope. tions was performed as described (3). For detection, Alexa Fluor-488, -594, or -405 (Invitrogen) Dissociation and FACS. To isolate DA neurons, astrocytes, and antibodies were used. For double immunohistofluorescence with oligodendrocytes, transgenic mice including TH-GFP (1), two antibodies raised in rabbit, Zenon Rabbit IgG Labeling Kits GFAP-GFP (Jackson Laboratories), and CNP-GFP mice (2) (Molecular Probes/Invitrogen) was used following the manufac- were used, respectively. For DA neurons and cortical or hip- turer’s instructions. Nuclei were visualized with DAPI. All pocampal astrocytes, P4-P8 pups were used. Oligodendrocytes images were collected with a confocal microscope (Leica TCS were collected by using P13/P20 animals. Solitary DA neurons SP2). were prepared as described by David Sulzer online protocol The following antibodies were used for this study: anti-Myc (http://www.sulzerlab.org/Sulzer_VM_culture_4.1.pdf). Briefly, (1:1,000; Cell Signaling), anti-Flag (1:1,000; Sigma), anti-mouse midbrain was isolated from transgenic TH-GFP mice. The pieces Hb (1:1,000; Cappel), chicken anti-mouse Hb (1:300; ICL), were enzymatically dissociated (Papain) under continuous oxy- anti-CNP (1:500; Chemicon), anti-TH (1:1,000; Sigma), anti- genation (5% CO2 and 95% O2 gas mixture) with slow stirring Gata-1 (1:200; SC), anti-Tal1 (1:200; SC), anti-GFAP (1:1,000; at 33 °C (0.5 M kynurenate is added to the solution). After 40 Sigma), anti-NeuN (1:1,000; Chemicon), anti-NG2 (1:300; min the reaction was stopped with enzyme inhibitors and the cell Chemicon), anti Iba-1 (1:200; kind gift of Dr. Yoshinori Imai), suspension was gently triturated with p1000 tip. Undissociated goat anti-human Hb (1:100; Biomeda), and mouse anti-human tissue was allowed to settle down, the supernatant was collected, Hb (1:200; Santa Cruz). Biagioli et al. www.pnas.org/cgi/content/short/0813216106 1of11 Postmortem Human Brain Samples. Brain samples were obtained noprecipitated proteins were eluted with 2 ϫ SDS sample buffer, from the brain banks of the Institute of Neuropathology, Bell- boiled, and analyzed by Western blot. Washes of immunopre- vitge Hospital, and the University of Barcelona. Human brains cipitated proteins were performed with the same immunopre- were obtained at autopsy with the informed consent of patients cipitation buffer. or their relatives and the institutional approval of the Ethics For Western blot analysis, samples were resolved on 10–15% Committee of the University of Barcelona. The samples were SDS/PAGE as needed and proteins were transferred to nitro- from three Caucasian males, age-matched (39–56 years old). The cellulose membrane (Schleicher & Schuell). Membrane was time between death and brain processing was between 3 and 5 h. blocked with 5% nonfat milk in TBS solution (TBS ϩ 0.1% SN and amygdala were excised, cryoprotected with 30% sucrose Tween20), then incubated with primary antibodies overnight at in 4% formaldehyde, frozen in dry ice, and stored at Ϫ80 °C until 4 °C or at room temperature for 2 h. Proteins were detected by use. horseradish peroxidase-conjugated secondary antibodies (Da- koCytomation) and enhanced chemiluminescence reagents (GE Constructs. Full-length mouse sequences of adult and embryonic Healthcare). globin chains and myoglobin and cytoglobin were obtained from the FANTOM2 RIKEN clone libraries collection (4). Mouse Microarray Analysis. Total RNA was isolated by using TRIzol neuroglobin full-length sequence was obtained from the German (Invitrogen) according to the manufacturer’s instructions, Science Centre for Genome Research, Berlin. Coding sequences treated with DnaseI (Ambion), and purified with a RNeasy Mini were subcloned by PCR into pIRES2-EGFP (Clontech/BD kit (Qiagen). RNA quality and quantity was assessed with an Biosciences) in frame with C-terminal myc tag. Agilent 2100 Bioanalyzer (Agilent Technologies) and NanoDrop We took advantage of pBudCE 4.1 vector (Invitrogen), de- 1000 spectrophotometer (Thermo Scientific). Two micrograms signed for the independent expression of two genes from a single of each total RNA sample was labeled according to standard plasmid, to express ␣- and ␤-chains of mouse Hb in MN9D cells. one-cycle amplification and labeling protocol (Affymetrix). A fragment composed by ␤-globin-Myc-tag-IRES-EGFP was Labeled cRNA was hybridized on Affymetrix GeneChip cloned into the CMV-MCS (SalI/XbaI) and 2ϫFlag-␣-globin Mouse Genome 430A 2.0 Arrays, containing 22,690 probesets was placed into EF-1 ␣-MCS (KpnI/XhoI). corresponding to Ϸ14,000 well-characterized mouse genes. Hy- A control vector containing only IRES-EGFP in CMV-MCS bridized arrays were stained, washed (GeneChip Fluidics Station (SalI/XhoI) was also created. 450), and scanned (GeneChip Scanner 3000 7G). Cell intensity values and probe detection calls were computed with Affymetrix Cell Culture and Transfection. HEK-293T cells were grown in GeneChip Operating Software.
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