Biotinylated anti species secondary antibodies

Embryonic brain sections were washed in PBS, blocked in a solution of 5% normal goat serum (Merck KGaA) (v/v) containing 0.1% Triton X-100 (v/v) (Merck KGaA) in PBS at RT for 2 h. They were first incubated in primary antibodies at RT for 2 h and, then, at 4 °C overnight. The following antibodies were used: mouse monoclonal Nestin (1:100, DSHB) and 5-Bromodeoxyuridine (BrdU; 1:1000, Progen), rat monoclonal anti-Ctip2 (1:500, Abcam), chicken polyclonal raised against GFP (1:500, Aves Laboratories), rabbit polyclonal raised against calbindin (CB-28; 1:3000, Swant), cleaved caspase-3 (CC3; 1:250, Cell Signalling Technology), Cux1 (1:100, Santa Cruz Biotechnology), Cdh13 (1:500, Millipore), L1 (L1; 1:1000, Millipore) or phospho-histone H-3 (PH-3; 1:1000, Millipore). Following incubation in primary antibodies, sections were washed in PBS, incubated in biotinylated anti-species secondary antibodies (1:250; Vector Laboratories) for 2 h and processed using conventional immunohistochemistry protocols described previously (Andrews et al. 2008 (link)).

For immunohistochemistry, muscle biopsies were fixed in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (PFA), made in PBS, for 4–8 h at room temperature (RT). Following fixation, tissues were cryoprotected in 30% sucrose in Diethyl Pyrocarbonate (DEPC) treated PBS, embedded and frozen in a mixture of 15% sucrose/50% Tissue-Tek OCT (Sakura Finetek), and sectioned in the coronal plane at 20 µm using a Cryostat (Bright Instruments). Patient muscle biopsies sections were washed in PBS, blocked in a solution of 5% normal goat serum (Merck KGaA) (v/v) containing 0.1% Triton X-100 (v/v) (Merck KGaA) in PBS at RT for 2 h. They were first incubated in primary antibodies at RT overnight. The following antibodies were used: pan-AKT (1:200, Cell Signaling Technology #2920), phospho-AKT (p-AKT; 1:200, Abcam #ab81283), S6 (1:200) and phospho-S6 (1:200). Following incubation in primary antibodies, sections were washed in PBS, incubated in biotinylated anti-species secondary antibodies (1:250; Vector Laboratories) for 2 h. Sections were washed and incubated with bisbenzimide (10 min in 2.5 μg/ml solution in PBS; Merck KGaA). Images were collected using an SP2 Leica confocal microscope (Leica Microsystems, UK). Sequential images were subsequently reconstructed using Metamorph imaging software (Universal Imaging Corporation, West Chester, PA).