Anti-CNTN2 (TAG-1) [IPI-CNTN2.57] 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.

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 for 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 (not shown): no primary control.