Table 1.
GenBank accession numbers and Real-Time PCR melting peak temperatures.
Figure 1.
Sequence Homology between Species.
Coding regions for BDNF (A) and Ntrk2 (C) were aligned between human, mouse, rat, pig, and horse using mVISTA to show inter-species similarities. Results are displayed as percent conservation between all species as compared to the human sequence. Phylogenetic trees were created for BDNF (B) and Ntrk2 (D) to visually illustrate which species were most closely related. bp: base pairs.
Table 2.
Comparison of the coding region of BDNF mRNA across species.
Table 3.
Comparison of the coding region of BDNF across species.
Table 4.
Comparison of the coding region of Ntrk2 mRNA across species.
Table 5.
Comparison of the coding region of Ntrk2 across species.
Figure 2.
Isolation of Uterine BDNF and Ntrk2 Transcripts.
Real-Time PCR melting peaks for uterine BDNF and Ntrk2 in human (A, B), mouse (C, D), rat (E, F), pig (G, H), and horse (I, J). Both primer pairs isolated specific products which were verified by sequencing in all species except for a non-specific peak (*) obtained with the Ntrk2 primers in pig uterus (H).
Figure 3.
Assessing Antibody Specificity.
Mouse brain sections were stained by immunohistochemistry with anti-BDNF (A) or Ntrk2 (D) antibodies as positive controls, or with antibody which had been pre-incubated with human recombinant BDNF (B) or Ntrk2 (E) protein, or with normal goat serum as a negative control (C, F). Decreased or absent staining was observed in pre-incubated sections as compared to positive controls (A vs. B; D vs. E). A 2X serial dilution of human recombinant BDNF (G) and truncated Ntrk2 (H) revealed bands of the appropriate sizes by Western Blot. Green: BDNF, brown: Ntrk2, blue: nucleus. Arrowheads: Purkinje cells, Gr: Granular layer, Mol: Molecular layer.
Figure 4.
Immunohistochemical localization of BDNF in the Uterus.
Uterine sections were stained for BDNF (A–F) using DAB as a chromogen (brown stain) or incubated with normal goat serum as a negative control (G–L). BDNF immunoreactivity was observed in human (A), mouse (B), rat (C), pig (D), horse (E), and bat (F) uterus. It localized to the luminal epithelium (LE), glandular epithelium (GE), smooth muscle of the myometrium (M) and vascular smooth muscle (vSM) in the mammals examined. Original image magnification was 200X. Scale bar represents 50 μm. L: lumen, S: stroma.
Figure 5.
Immunohistochemical localization of Ntrk2 in the Uterus.
Uterine sections were stained for Ntrk2 (A–F) using DAB as a chromogen (brown stain) or incubated with normal goat serum as a negative control (G–L). Ntrk2 immunoreactivity was observed in human (A), mouse (B), rat (C), pig (D), horse (E), and bat (F) uterus. It localized to the same areas as its ligand BDNF. Ntrk2 was observed in the luminal epithelium (LE), glandular epithelium (GE), smooth muscle of the myometrium (M) and vascular smooth muscle (vSM) in the mammals examined. Original image magnification was 200X. Scale bar represents 50 μm. L: lumen, S: stroma.
Figure 6.
BDNF and Ntrk2 Expression in the Human Uterus.
Uterine homogenates were collected from hysterectomy patients and probed for BDNF (A) and Ntrk2 (B) by Western Blot, using mouse brain as a positive control. Uterine samples were loaded in lanes 1–8, and mouse brain homogenate in lane 9.