Anti-NRP1 [IPI-mNRP1.23] in immunohistochemistry (IHC) (Mouse)

Immunohistochemistry Methods: Mouse embryonic spinal cord

Mouse embryos were first purchased from Jackson Labs. Instructions were provided to Jackson labs for harvesting and fixing tissue. 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 minutes in 1X PBS, then stored in 1X PBS + 0.01% sodium azide 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.

Equilibrated tissue was then frozen in Optimum Cutting Temperature (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: commissural axon-labeling counterstain TAG1.

Negative control: No primary control.

References

Himmels et al. (2017). Motor neurons control blood vessel patterning in the developing spinal cord. Nat. Commun. 814583. https://doi.org/10.1038/ncomms14583 (Link opens in a new window) PMID: 28262664 (Link opens in a new window)