Anti-SEMA7A [IPI-mSEM7A.19] in immunohistochemistry (IHC) (Mouse)

Immunohistochemistry Methods: Mouse brain tissue

Adult (3–5 week old) mouse brains were first purchased from Jackson Labs. Instructions were provided to Jackson labs for harvesting and fixing tissue. 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 (NaN₃) at 4 °C. Upon arrival of tissue, 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 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: NeuN counterstain (anti-mouse IGG1).

Negative control: No rabbit primary antibody control.

Observations

Signal is faint but appears specific to the molecular layer of the cerebellum and Purkinje cell bodies, consistent with literature showing high transcript levels of Sema7a in Purkinje neurons throughout mouse development.

References

Uesaka et al. (2014). Retrograde semaphorin signaling regulates synapse elimination in the developing mouse brain. Nat. Commun. 814583. https://doi.org/10.1126/science.1252514 (Link opens in a new window) PMID: 24831527 (Link opens in a new window)