Subplate neuron ablation alters neurotrophin expression and ocular dominance column formation -- Data Supplement - Supplemental Methods -- Proceedings of the National Academy of Sciences

Supplementary material for Lein et al. (1999) Proc. Natl. Acad. Sci. USA 96 (23), 13491-13495.

Materials and Methods
ISEL Staining Methods.
A modified version of the in situ end-labeling (ISEL+) method of Blaschke and Chun (1) was used. Thin (12-mm) sections were cut by cryostat, dried under vacuum, fixed with 4% paraformaldehyde for 30 min, extracted with 0.6% Triton X-100 for 30 min, washed, acetylated, and washed again (washes in 2´ sodium chloride/sodium phosphate/EDTA). Endogenous peroxidase was then quenched in 3% H₂O₂ for 5 min, and sections were finally washed and dehydrated through graded alcohols. A reaction mixture containing 1 mM biotin-dUTP (Boehringer Mannheim), 0.15 units/ml terminal transferase (TdT; GIBCO), 1´ TdT buffer, and 1% bovine serum albumin was applied, and sections were incubated for 1 hr at 37°C. Sections were washed and then incubated in NeutraLite avidin–horseradish peroxidase (Molecular Probes) at a dilution of 1:1,000 in NEN block for 30 min. After final washes, signal was developed with tyramide signal amplification (TSA)-Direct Cy3 (NEN). Sections were counterstained with TOPRO3 (Molecular Probes) and analyzed on a Leica scanning confocal microscope. Control sections from mouse thymus and liver were used to test the sensitivity of the ISEL method (2). BrdU Staining.
Timed pregnant cats were given intravenous injections of 100 mg/kg BrdU (Sigma) between embryonic day 24 and 26 to label subplate neurons. BrdU-labeled subplate neurons were visualized by using a modification of the method of Dinjens (3). Sections were processed as described for the ISEL procedure. To expose the incorporated BrdU, sections were microwaved for 10 min in 0.1 M sodium citrate, pH 5. Anti-BrdU antibody (Caltag, Burlingame, CA) was applied at 1:25,000 with 100 units/µl ExoIII (Roche Molecular Biochemicals) in ExoIII buffer plus 100 mM NaCl and 1% bovine serum albumin, and sections were incubated at 37°C for 1 hr. After washing, horseradish peroxidase-conjugated anti-mouse secondary Ab (Jackson ImmunoResearch) was applied at a dilution of 1:200. Sections were washed, amplified with TSA Direct (FITC; NEN) per manufacturer's instructions, and counterstained with TOPRO3.

1. Blaschke, A. J., Staley, K. & Chun, J. (1996) Development (Cambridge, U.K.) 122, 1165-1174.

2. Schwartz, L. M. & Osborne, B. A. (1995) in Methods in Cell Biology, eds. Wilson, L. & Matsudaira, P. (Academic, San Diego), Vol. 46, Chap. 2.

3. Dinjens, W. N., ten Kate, J., Lenders, M. H., van der Linden, E. P. & Bosman, F. T. (1992) Histochemistry 98, 199-205.