Tissue tek 2

For imaging, SeeDB-cleared whole brain samples were put into a Tissue-TEK II (No. 2, Sakura Finetek, Japan) and covered by a micro cover glass (24 × 60 mm, thickness No.1 0.12–0.17 mm, Matsunami, Japan). The SeeDB solution was used for immersion (Supplementary Fig. 4). Imaging was performed using a LSM 7 MP multiphoton microscope (Zeiss) with a HighQ-2 laser (Spectra-Physics), a BiG detector, and a 20× objective lens (1.05 NA) from the dorsal cortical surface. RFP in callosal projection neuron were excited, and emitted fluorescence was filtered (575–620 nm). Sequential z-images consisted of optical sections (512 × 512 pixels; 1.19 μm/pixels) with 1.74 μm intervals. Laser power was manually adjusted to give constant fluorescent intensities at all depths. The whole cellular morphology of single L2/3 callosal projection neuron was reconstructed from totally 23 × 1 blocks (1block = 607.28 μm × 607.28 μm, Z_max = 1.5 mm from the surface) images from the electroplated side to contralateral side where the axons projected. Acquired three-dimensional images were analyzed using the IMARIS Filament Tracer software (Bitplane).

To clarify the effect of MSTN knockout on skeletal muscle, we performed histological analysis and statistical quantitative analyses of muscle fibers in skeletal muscle of mutant and wild-type channel catfish. Four each of one-month-old fry from mutant (individuals with frame-shift mutation) and control groups were euthanized with tricaine methane sulfonate (MS-222) (Western Chemical Inc., Ferndale, WA). Subsequently, catfish muscles were dissected, cross sectioned and fixed in 4% paraformaldehyde at room temperature for at least 24 hours, and then dehydrated and paraffin embedded using Tissue Tek II^® (Sakura Finetek USA, INC, CA). Serial sections were made at 7 μm thickness using a rotary microtome (American Optical Corporation, Southbridge, MA). The sections were mounted on glass slides and stained with the regressive staining method using Harris hematoxylin (VWR International, PA) and eosin with phloxine (Sigma-Aldrich, Inc., MO). Muscle fibers were counted. Cell numbers were calculated as the number of fibers per cross-sectional muscle area using the “Cell Counter” features of ImageJ program^{84 (link)} and used for evaluating fiber size.