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Figures
- Fig. 1
(A) Example of a Nissl-stained section taken with Nikon Eclipse 80i Microscope with good section quality. Scale = 44 μm. (B) Example of a section labelled for NeuN (green). Image was taken with a Nikon Eclipse 80i Microscope. Scale = 58 μm. (C) Example of a section labelled for dopamine transporter (DAT; green). Image was taken with a confocal laser scanning microscope (CLSM). The inset is an example of a section labelled with only the secondary antibody, demonstrating minimal labelling, and therefore specificity of the secondary antibody. (D) Example of a section labelled with the tyrosine hydroxylase (TH) antibody (green) taken with a CLSM. The inset is an example of a section labelled with the primary antibody for 3-nitrotyrosine (3NT), and secondary antibody for TH. Minimal labelling was observed, indicating specificity of the secondary antibody. (E) Example of a Förster resonance energy transfer (FRET) experiment for DAT and 7-diethylamino-3-(4′maleimidylphenyl)-4-methylcoumarin (CPM) FRET. The red circle indicates the area that has been photobleached. Increase in the intensity of the donor fluorophore (blue; CPM) can be observed after bleaching the acceptor fluorophore (Alexa488; green), indicating the presence of FRET. (F) Example of a FRET experiment for TH and 3NT FRET. The red circle indicates the photobleached area. Increase in the intensity of the donor fluorophore (Alexa350; blue) can be observed after photobleaching the acceptor fluorophore (Alexa488; green), indicating presence of FRET.
- Fig. 2
(A) Correlation between ΔIF for dopamine transporter (DAT) and 7-Diethylamino-3-(4′maleimidylphenyl)-4-methylcoumarin (CPM) and brain pH, age and postmortem interval (PMI). (B) Correlation between ΔIF for tyrosine hydroxylase (TH) and 3-nitrotyrosine (3NT) and brain pH, age and PMI. Results were assessed using the Pearson correlation coefficient. The correlations were not significant.
- Fig. 3
(A) Intensity of 7-Diethylamino-3-(4′maleimidylphenyl)-4-methylcoumarin (CPM) labelling after correction for nonspecific CPM binding with negative control (sections treated with iodoacetamide and N-ethylmaleimide). Results are expressed as mean ± standard error of the mean (SEM). *p < 0.05; **p < 0.01. n = 50 (control, n = 13; bipolar disorder [BD], n = 13; schizophrenia [SCZ] = 11, major depressive disorder [MDD] = 13). (B) 3NT immunoreactivity after correction for nonspecific signals as measured by fluorescence intensity. Results are expressed as mean ± SEM, n = 50 (control = 14, BD = 10, SCZ = 13, MDD = 13). All analyses were performed on nonprocessed, original images in original imaging format (OIF).
- Fig. 4
(A) Change in fluorescence (ΔIF) between Alexa488 labelling dopamine transporter (DAT) and CPM labelling free thiols. Higher values indicate greater amounts of free thiols in DAT-IR regions after correction for background ΔIF (FRET in oxidized sections). Results are expressed as mean ± SEM *p < 0.05 **p < 0.01. n = 54 (Control = 14, bipolar disorder [BD] = 14; schizophrenia [SCZ] n = 12; major depressive disorder [MDD] n = 14). (B) ΔIF between Alexa488 labelling tyrosine hydroxylase (TH), and Alexa350 labelling 3-nitrotyrosine (3NT). Greater ΔIF indicates greater nitration. Results are expressed as mean ± SEM. *p < 0.05; **p < 0.01. n = 50 (Control n = 11, BD n = 11, SCZ n = 14, MDD n = 14). All analyses were performed on nonprocessed, original images in Original Imaging Format (OIF).
Tables
Group; mean (range)* Characteristic Control Bipolar disorder Schizophrenia MDD Age, yr 48 (29–68) 42 (25–61) 44.2 (25–62) 46.4 (30–65) PMI, h 24 (8–42) 33 (13–62) 34 (12–61) 28 (7–47) pH 6.3 (5.8–6.6) 6.2 (5.8–6.5) 6.1 (5.8–6.6) 6.2 (5.6–6.5) Sex, male:female 9:6 9:6 9:6 9:6 Lithium, no. 0 4 2 2 Antipsychotics, no. 0 8 12 0 Antidepressants, no. 0 8 5 10 MDD = major depressive disorder; PMI = post mortem interval.
↵* Unless otherwise indicated.
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