3% Triton X-100 and 1% normal goat serum (NGS) in 0.1 m PBS for 24 h at 4 °C. After rinsing three times for 30 min in 1 × PBS at room temperature, the tissue was incubated with secondary antibodies for 2 h at room temperature. Slices were again washed three times for 45 min in 1 × PBS. Sections were counterstained with DAPI (1 : 10 000), washed in PBS and eventually mounted in Moviol on glass slides. In control experiments, immunostaining was performed with all but the primary antibodies. No specific staining was observed under these conditions. For control of Reelin immunoreactivity, staining was performed in addition in Obeticholic Acid clinical trial tissue from Reelin-deficient reeler mutants (see Fig. 5B).
SPNs undergo a complex migration process which in the case of wild-type mice comes to an end at the intermediolateral column (IMLC). Histone Methyltransferase inhibitor In
contrast, in reeler mice the migration of SPNs continues towards the central canal. It has been proven useful to determine this additional migration in reeler mutants by dividing the distance from the IMLC to the central canal into three segments (segment A = region of IMLC, segment B = intermediate region, segment C = region of central canal; see Yip et al., 2009). To compare the migration of SPNs in the different genotypes, their actual location along a line from the lateral edge of the IMLC to the central canal was determined using Imagej software (National Institute of Health). As individual sections of the spinal cord differ in size, the measured distance from the IMLC was divided by the total distance from the IMLC to the central canal to obtain a percentage value. For these measurements, all 50-μm
slices were optically cut into 4-μm slices and photographed using a Zeiss LSM 510 NLO spectral confocal Afatinib microscope. All SPNs were counted in the four different genotypes (wild-type animals, reeler mutants, apoer2 knockout mice, vldlr knockout mice), and their distribution in the three segments was determined. Wild-type embryos (E13.5; n = 6) and reeler embryos (E13.5; n = 6) were harvested from pregnant, anesthetized dams (i.p. injection of 10 mL/kg Avertin; Sigma). The tails were used for genotyping. The spinal cord was removed, and thoracic and lumbar levels were divided in the midline. One side was treated with Reelin-containing supernatant, while the other was treated with Mock-control supernatant (Förster et al., 2002; Chai et al., 2009). For this, the tissue was stored in ice-cold Hank’s buffered salt solution (HBSS; Invitrogen) before it was chopped into small pieces with a tissue chopper. The tissue lysate was then collected into 1.5-mL tubes and resuspended in ice-cold HBSS. After centrifugation, the buffer was discarded, and 1 mL of Reelin-containing supernatant or Mock control supernatant was added and resuspended. Tissue lysates were then incubated for 20 min at 37 °C.