Anti-UNC5A [IPI-mUNC5A.50] in immunohistochemistry (IHC) (mouse)

Immunohistochemistry Methods: Mouse embryonic spinal cord or brain tissue:

Mouse embryos or adult mouse brains were first purchased from Jackson Labs. Instructions were provided to Jackson labs for harvesting and fixing tissue. For embryos: immediately after harvesting by C-section, embryos were fixed in 4% PFA in 1X PBS for 2 h at 4 °C, then washed 3x 10 min in 1X PBS, then stored in 1X PBS + 0.01% sodium azide (NaN₃) at 4 °C. For adult (3–5 week old) brains: anesthesia was performed with inhalant isoflurane, followed by transcardial perfusion with ~11 mL 1X PBS perfusate/mouse containing heparin (10,000 units heparin per 1L 1X PBS), then ~25 mL ice-cold 4% paraformaldehyde in 1X PBS. After dissecting the brain, it was stored in 1X PBS + 0.01% sodium azide solution at 4 °C. Upon arrival of tissue, embryos were washed 3x 10 min in 1X PBS, then cryoprotected overnight in 30% sucrose in 1X PBS, at 4 °C. Adult brains were placed in a 5 mL conical tube containing ice-cold (4 °C) 10% sucrose solution and incubated at 4 °C overnight. The next day, 10% sucrose was replaced with ice-cold 30% sucrose and the brain was allowed to equilibrate completely at 4 °C, as evidenced by its sinking to the bottom of the tube, for 2–3 days.

Equilibrated tissue was then frozen in OCT on dry ice and 20–25 μm sections were cut on a Leica cryostat at the Harvard histology / sectioning core. Slides were thawed for 5 min at RT; sections were rehydrated 1x 5min in 1X PBS and then washed 3x 5 min in 1X PBST (0.1% Triton X-100 in 1X PBS) in a humidified chamber in a slide box. Slides were blocked for 1 h at RT in 5% NGS (Normal Goat Serum) or 5% NDS (Normal Donkey Serum) in 0.1% Triton-X-100 in PBS. Primary antibodies were then diluted in blocking solution and incubated overnight at 4 °C Slides were then washed 4x 5min in 0.1% TritonX-100 and incubated with secondary antibodies diluted in blocking solution (5% NGS or NDS) at RT for 1.5–2 h, in the dark. Slides were washed 4x 5 min in 0.1% TritonX-100 again, then 1x 5min in 1X PBS. Slides were then mounted in Fluoromount mounting medium with DAPI, outlined with nail polish to secure the coverslip, and stored at 4 °C in the dark until ready to image.

Controls

Positive control: NeuN (hippocampus) or TAG-1 (spinal cord) counterstain (anti-mouse IGG1).

Negative control: No primary control.

Observations

In the adult brain, this antibody shows labeling in the CA1, CA3, and dentate gyrus (DG) regions of the hippocampus above background levels, which matches the expected localization pattern of UNC5A protein based on the expression pattern of Unc5a mRNA in the adult hippocampus (Allen Brain Atlas (Link opens in a new window)). Neurites are detectable and the antibody labels neurons in a mosaic manner in these sections.

In the mouse embryonic spinal cord, the antibody labeling is enriched in the ventral spinal cord, demonstrating strong signal in the lateral funiculus and gray matter but not on the commissural axons or the dorsal root ganglia, consistent with literature detailing the localization of UNC5A protein in the mouse embryonic spinal cord (Williams et al., 2006).

References

Williams et al. (2006). UNC5A promotes neuronal apoptosis during spinal cord development independent of netrin-1. Nat Neurosci. 9(8):996-8. https://doi.org/10.1038/nn1736 (Link opens in a new window) PMID: 16829956 (Link opens in a new window)