PROTOCOLS

Immunohistochemistry at E18.5

In 19% of lethal lines, embryos appear morphologically normal at E14.5. The neural tissue in the brain and spinal cord of embryos from these lines are examined again at E18.5 using immunohistochemistry.

Sample preparation

The brain is dissected from the skull and stored with the body in 4% paraformaldehyde (PFA) at 4C. The body is divided into three (cervical, thoracic and lumbar sections) and placed in sucrose solution at 15% and 30% for 24 hours at each concentration.

Samples are placed in Optimum Cutting Temperature (OCT) compound and frozen in dry ice, before storing at -80C. A cryostat is used to cut 20 μm coronal sections through the entire brain, and 20 μm transverse sections through the entire cervical, thoratic and lumbar sections.

Note that if more than two embryos are available, two brains are cut into coronal sections and the others are cut into sagittal sections (also 20 μm).

Staining

Two stains are used on the sections prior to imaging:

  1. Calretinin + Neurofilament
    This combined stain highlights interneurons and axons.

    • A blocking solution of 5% goat serum and PBST (0.5%) is mixed. This is left on the slide for 1 hour at room temperature.
    • The primary antibodies Calretinin and Neurofilament are mixed with the blocking solution at the concentrations: Calretinin 1:1000; Neurofilament 1:50. This solution is left on the slide overnight at 4C.
    • The slide is washed with PBS.
    • Secondary antibodies goat anti-rabbit(488) and goat anti-mouse (564) are mixed with the blocking solution in the ratio 1:1:400. This solution is left on the slide for 2 hours at room temperature.
    • The slide is washed with PBS.
    • A solution of hoechst stain and PBS is mixed in the ratio 1: 2000. This is left on the slide for 5 minutes at room temperature.
    • The slide is washed with PBS and mounted using Fluorsave.
  2. Nissl
    This stain highlights neural death.

    • A solution of Na acetate (13.6g/l) and acetic acid (6ml/L) is mixed in the ratio 2.5:1. Thionin (1%) is then added in the ratio 1:40.
    • The slide is stained in this solution for 5 minutes.
    • The slide is submerged in a gradient ethanol solution (70, 80, 95, 100%) for 1 – 2 minutes at each concentration.
    • The slide is submerged in histoclear solution for 15 – 30 seconds before drying and mounting using limonene.

Analysis

The slides are scanned using a virtual microscopy scanning system. The intensity and distribution of the staining is then analysed to identify developmental abnormalities in the brain and spinal cord.

Immunohistochemistry at E18.5 is performed by the Houart Lab at King’s College London.