Effects might complicate the interpretationVitamin B12 and ParkinsonFigure four. Expression of transcobalamin II/oleosin (TCII/OLEO) chimeric proteins in rats 60 days following transfection using the NTSpolyplex. A: RT-PCR from the plasmid transcripts inside the substantia nigra of rats. A group of rats (n = three) was transfected with the plasmid Foliglurax Cancer pCMV-TCII-OLEO and yet another (n = three) together with the plasmid pCMV-OLEO-TCII. RT-PCR amplified a fragment of 380 bp for TCII-OLEO, a fragment of 394 for OLEO-TCII, and also a fragment of 349 for b-actin, the internal handle. Lane 1 corresponds to the amplified fragment in the plasmid (constructive handle). Lane two is actually a PCR in the absence of plasmid or cDNA (adverse handle). The amplified item in the transfected substantia nigra of each rat corresponds to the lanes three, five, and 7, along with the lanes four, six, and eight show the RT-PCR outcome from the non-transfected side. B: GFP immunofluorescence within the rat substantia nigra transfected with pCMV-GFP-TCII-OLEO. The pCMV-GFP-TCII-OLEO encodes for the fusion protein green fluorescent protein-transcobalamin-oleosin (GFP-TCII-OLEO). The immunofluorescence was completed with a mouse monoclonal antibody to GFP as well as a donkey antimouse IgG fluorescein labeled. Representative micrographs of coronal section of manage substantia nigra (1) and transfected substantia nigra (two) with the very same rat are presented. Calibration bars = one hundred mm. C: Double immunofluorescence against TCII and tyrosine hydroxylase (TH) in the substantia nigra of rats. The neurons had been transfected with NTS-polyplex with pCMV-TCII-OLEO coding for transcobalamin-oleosin (TCII-OLEO). Slices from mesencephalon (40 mm) were immunostained at 7-day just after transfection. The principal antibodies had been a goat polyclonal anti-TCII and a mouse monoclonal anti-TH. The secondary antibodies had been a donkey Picloram MedChemExpress antigoat IgG fluorescein labeled and also a donkey antimouse IgG rhodamine labeled. Representative micrographs of coronal section of handle substantia nigra (1) and transfected substantia nigra (four) on the similar rat are presented. Calibration bars = 50 mm. doi:10.1371/journal.pone.0008268.gPLoS 1 | plosone.orgVitamin B12 and ParkinsonFigure five. Apoptosis of tyrosine hydroxylase (TH) immunoreactive cells inside the substantia nigra of rats transfected with a number of plasmids. A: TH-immunoreactive neurons immediately after transfection. The neurons were transfected with NTS-polyplex with among the list of following plasmids, pCMV-TCII-OLEO coding for transcobalamin-oleosin (TCII-OLEO, 1), pCMV-OLEO-TCII coding for oleosin-transcobalamin (OLEO-TCII, 2), pCMV-TCII coding for transcobalamin II (TCII, three), pCMV-OLEO coding for oleosin (OLEO, 4), as well as the pCDNA3, the empty plasmid (five). Mesencephalon slices (40 mm) had been immunostained at 2-month after transfection using a mouse monoclonal antibody to TH and a donkey antimouse IgG fluorescein labeled. Representative micrographs of sagital section with the rat mesencephalon are presented. Calibration bars = 200 mm. B: Apoptosis in THimmunoreactive neurons after transfection with the plasmid pCMV-TCII-OLEO. Representative micrographs of the substantia nigra (with double immunostaining at 15-day following transfection) are presented. The principal antibodies have been a mouse monoclonal antibody to TH, and a rabbit polyclonal antibody to cleaved Caspase-3. The secondary antibodies included a donkey anti-mouse IgG FITC labeled (1 and 4), and also a donkey antirabbit IgG rhodamine labeled (two and 5). Representative micrographs of coronal section of control substantia n.