M205 fa

The indexes of the liver, spleen, and thymus were calculated based on the following formula: total weight of mouse liver or spleen or thymus (mg)/total weight of mouse (g). The area of egg granulomas was measured on H&E-stained tissue sections using Image Pro Plus 6.0 software. The area of each granuloma in H&E-stained sections was measured. Liver tissues were weighed and digested overnight in 4% potassium hydroxide, and released eggs were then counted. Worms were fixed with 95% ethanol, 3% formalin, and 2% glacial acetic acid and then stained with 2.5% hydrochloric carmine red (Merck) for 1 h and preserved on glass slides. The worms were observed under a stereomicroscope (Leica M205FA, Germany), and worm length was calculated using the stereomicroscope (Leica M205FA). Fibrosis was detected by staining liver sections with Masson’s trichrome.

Animals were anesthetized using L-menthol based on a previously reported method^{19 (link),25 (link)}. In brief, 0.56% (weight per volume) L-menthol in ethanol was prepared and used as a stock solution. The stock solution was further diluted with 1% (volume per volume) artificial seawater before use. Animals were soaked in the diluted L-menthol solution for 10 min. Part of the tunic, body-wall muscle, and pharynx were excised under a fluorescence stereomicroscope (Leica M205 FA; Leica Microsystems, Wetzlar, Germany), and the whole animals were fixed in 4% paraformaldehyde in PBS at 4 °C overnight. The fixed specimens were soaked in 2 mg/ml glycine in PBS to quench the paraformaldehyde. The specimens were then washed three times with PBS and used for morphological analyses. Anatomic analyses were performed under the stereomicroscope (Leica M205 FA; Leica Microsystems) using precision tweezers (Outils Rubis SA, Stabio, Switzerland) and microscissors (Inami & Co., Ltd., Tokyo, Japan).

The culture method has been described previously^{7 (link),34 (link),35 (link)}. Briefly, the testes of rats, postnatal days 3–9 (P3–P9), were decapsulated and gently separated by forceps into 20 to 40 pieces per testis. The tissue fragments were then placed on a block of 1.5% agarose gel half-soaked in the well of a 12 well-culture plate containing 0.5 mL of culture medium. Before culture, the gel block was submerged twice in medium, for more than 6 h each time, with a medium change in between. Each tissue piece on the agarose gel block was 1–3 mm in diameter and around 200 µm in thickness. Each gel block was loaded with 3 to 4 tissues. In cases in which the PC method was applied, a single tissue was placed on a gel, and a PC chip with an indentation depth of 160 µm was used to cover the tissue. The PC was prepared as described in previous papers^{32 (link),52 (link)}. The medium was changed once a week. The culture incubator was supplied with 5% carbon dioxide and 15% O₂ and maintained at 34 °C. Cultured tissues were observed once a week under a stereomicroscope equipped with an excitation light for GFP (LeicaM205 FA; Leica, Wetzlar, Germany). The extent of GFP expression in each tissue sample was assessed on a scale of 0–100% (0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100%), judging visually the ratio of the area of GFP expression in the whole region of each tissue fragment.

Tissues in culture were observed once a week under a stereomicroscope (Leica M 205 FA; Leica). The proportion of the area showing GFP expression was recorded to evaluate the efficiency of spermatogenesis. It was classified into 6 grades, 0‐5, based on the expression area: 0, ~20, ~40, ~60, ~80, and‐100%, respectively. This GFP grading scale faithfully corresponded to spermatogenic progression, as confirmed in previous studies.7, 12 To identify haploid cells with an acrosome cap structure, cultured tissues were observed with an inverted microscope applying the GFP‐excitation light (Olympus IX 73; Olympus).

The images of in situ hybridization, HE staining and SA-β-gal assay were captured using a Nikon ECLIPSE Ni microscope (Nikon) or Olympus microscope (IX83, Olympus). Immunostaining images were acquired using a Zeiss Axio Observer.Z1 microscope (Zeiss) or Nikon A1 confocal microscope (Nikon). Bright-field images of whole mount zebrafish embryos were taken using a Leica microscope (Leica M205FA, Leica).

Gemmae isolated from gemma cups located at the most basal position in thalli were stained by 15 μM propidium iodide and 0.01% Triton X-100 for 15 min and observed under the binocular fluorescent microscope (Leica M205FA, Leica, Wetzlar, Germany) using the DsRED filter set. The number of gemmae with elongated rhizoids were counted. A total of 60–114 gemmae were used for each data point of three biological replicates.

For live imaging, larvae were anaesthesized with ether, mounted in halocarbon 700 or 50% glycerol and observed either by confocal laser scanning microscopy (CLSM, using Zeiss LSM 710, 780 and 880) or fluorescent binocular Leica M205 FA.
For imaging of the cuticle preparations, the larvae were mounted in Hoyer's medium (30 g gum arabic, 50 ml distilled water, 200 g chloral hydrate, 20 g glycerol, mixed 1 : 1 with lactic acid), kept overnight at 65°C and observed on a binocular Leica M205 FA. Transmission electron microscopy was performed following our extensive protocol published in 2010 [23 ]. For examination of the cuticular ridges, third instar larvae were digested in Hoyer's medium and their cuticles washed several times in distilled water. Afterwards they were stacked to the metal plate and their inner side was scanned by the atomic force microscope (Innova AFM, Bruker).
For figures’ preparation Adobe Photoshop CS3/CS6 and Adobe Illustrator CS4/CS6 software were used without changing initial microscope settings. For cell measurements, AxioVision Rel. 4.7 was used.