N6876

Frzb in situ hybridization probe was kindly provided by Prof. De Robertis (Leyns et al., 1997 (link)). The labeled probe was ethanol-precipitated, resuspended in 100 mM DTT, diluted in hybridization solution (60% deionized formamide, 20 mM Tris-HCl, 5 mM EDTA, pH 8, 0.3 M NaCl, 0.5 mg/ml yeast RNA, 5% dextran sulfate). In situ hybridization was performed according to standard procedures (Mitsiadis et al., 1995 (link)). Briefly, slides were incubated with the probe at 60°C. After intense washing, the slides were incubated in blocking solution (20% Normal Goat Serum) and anti-digoxigenin (DIG)-AP (alkaline phosphatase conjugate) Fab-fragment (Boehringer Mannheim, 1093 274) diluted 1:1,000 in blocking solution. The color reaction was developed using Nitro Blue Tetrazolium (NBT, Sigma N-6876) and 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP, Sigma B-8503) in staining solution 2% NaCl, 5% MgCl2, 10% Tris-HCl pH 9.5, 1% Tween-20. In situ hybridization immediately followed by BrdU immunohistochemistry was performed in cryosectioned slides of E13–E15 mouse embryos to show the correlation between Frzb expression and cell proliferation (Mitsiadis et al., 2008 (link)). No hybridization signal was detected with the sense probe at these developmental stages.

Slides were thawed to room temperature for 20 min, and incubated in 0.05% nitrotetrazolium blue chloride (N6876, Sigma-Aldrich), 0.1 M sodium succinate (S2378, Sigma-Aldrich), and 0.01 M PBS at 37 °C for 40 min with gentle rocking. Sections were dehydrated in a series of graded ethanols, cleared by xylene, and cover-slipped by a solvent-based mounting medium (8310-16, Thermo Scientific).

O₂^.− was localized by staining with nitrotetrazolium blue chloride (NBT, N6876, Sigma)18 (link). The 3-cm-long subsamples (five per treatment) were immersed in HEPES-NaOH buffer (pH 7.6) containing 0.5 mg of NBT/ml and 10 mM NaN₃. The subsamples were vacuum infiltrated in this NBT solution for 30 min and were then held at room temperature until the blue colour (NBT-O₂^.−) became visible. The NBT-stained roots were photographed with a digital camera (DSC-F717, Sony, Japan) before semi-thin transverse sections (8 μm thick) were prepared. Semi-thin section was conducted by fixing aerial root samples in 0.1 M phosphate buffer (pH 7.2) containing 2% glutaraldehyde and 2.5% Paraformaldehyde. After 6 times wash with 0.1 M phosphate buffer, they were dehydrated by alcohol steeply and eddied in flat molds using EPON812 resin. Sctions (2 μm) were cut by ultramicrotome (Leica, UC6, Germany). The sections were observed and photographed with a light microscope (AX70, Olympus, Japan) and a digital camera (DP50, Olympus, Japan).

NADH-TR (diaphorase): Muscle sections were incubated in NADH-tetrazolium solution (NADH, Sigma N8129; Nitro blue tetrazolium, Sigma N6876) for 30 min at 37 °C. Unbound solution was removed from muscle sections using by successive washes with 30% acetone, 60% acetone, 90% acetone, 60% acetone, and 30% acetone. Sections were mounted with CitriSolve. Images were taken at 10X using an EVOS M7000 microscope. Intensity of NADH staining and cross-sectional area were analyzed in ImageJ by manually selecting each muscle fiber and recording mean intensity and area. 3 images were analyzed ( $\ge$ 574 fibers) per muscle for each genotype.
Androgen Receptor: Muscle sections of gastrocnemius from one knock-in mouse and one transgenic mouse were fixed in 4% PFA for 20 min, and stained as in Montie et al. [46 (link)]. Images were taken at consistent exposure on an EVOS M7000 microscope at 60x. 15 images per muscle were evaluated in ImageJ by selecting a region of interest for each muscle fiber and measuring mean intensity of AR fluorescence. All images were taken at the same exposure.