Xylol

Offspring from genetic families of H. huso, A. ruthenus and A. oxyrinchus were sacrificed by an overdose of buffered tricaine methanesulfonate (MS222; 0.3 g l⁻¹, PHARMAQ); individual length and wet weight were recorded, finclips (stored in 100% ethanol) and gonadal samples were taken immediately. The right gonad strand was fixed in phosphate-buffered formaldehyde solution (ROTI^®Histofix, 4%, Carl Roth) for at least 24 h at room temperature, then rinsed three times for 24 h with 70% ethanol, and stored at room temperature. Gonad strands were dehydrated in an increasing series of ethanol (75%, 90%, 95%, 100%), rinsed in xylol (Carl Roth) and transferred into Paraplast^® (Leica), using the Shandon Excelsior ES Tissue Processor (Thermo Fisher Scientific). Gonads were embedded in a spiral-like arrangement, to ideally cut the whole organ into 5 µm sections across its entire length (microtomes: Leica 2065 or Microm HM 340E, Thermo Fisher Scientific). Sections were mounted on glass slides and stained using standard haematoxylin/eosin (0.1%, Carl Roth). Histological evaluation was made at various magnifications using light microscope Nikon Eclipse Ni-U with Nikon DS-Fi3 camera, and the corresponding software Nikon DS-L4 to archive images.

All used materials and chemicals have been purchased in highest available purity
unless otherwise stated. Pre-cut silicon wafers
(10 × 10 mm²)
with 5 nm SiO₂ were kindly provided by AMS AG
(Unterpremstätten, Austria). Glass vials (4 mL,
Ø 15 mm, Rotilabo), microscopy slides, 2-propanol,
ethanol (analytical grade, not denatured) and xylol were purchased from Carl
Roth (Karlsruhe, Germany). Trimethylsilyl-cellulose (TMSC,
DP = 2.8) was purchased from Thüringisches
Institut für Textil- und Kunstoff-Forschung (TITK e.V, Germany).

Injected oocytes were fixed in 4% PFA (16% paraformaldehyde, E15700, Science Service, Munich, Germany) for 4 h at room temperature followed by dehydration gradient from 70% ethanol to xylol (Carl Roth, Karlsruhe, Germany) within 48 h. Samples were embedded in paraffin and cross-sectioned (5 μm) by using a Leica RM 2245 microtome (Leica Microsystems Heidelberg, Germany). Shortly before immunohistochemical treatment, paraffin was removed via xylol to ethanol gradient. Non-specific binding sites were blocked using 5% goat serum in phosphate-buffered saline and incubated with the same primary antibodies as for immunoblotting. Samples were incubated with the secondary antibodies Alexa Fluor-488 goat anti-rabbit and Alexa Fluor-594 goat anti-mouse (Life Technologies, Carlsbad, CA, United States) and examined by confocal laser-scanning immunofluorescence microscopy (LSM 710, Zeiss, Oberkochen, Germany).

Samples of femur and vertebrae L2 were fixed in 4% phosphate buffered paraformaldehyde (Carl Roth, Karlsruhe, Germany) and demineralized in 10% ethylene diamine tetra acetic acid (Merck, Darmstadt, Germany) in 0.281 M Tris-buffer. The samples were dehydrated using increasing ethanol concentrations (70%, 80%, and 96% each for 2 h, 3x 100% for 3 h) and incubated in xylol (Carl Roth, 3x 1 h) and afterwards in liquid paraffin. After blocking out, paraffin sections were cut with a thickness of 4-5 μm at the rotation microtome (RM 2155, Leica, Bensheim, Germany). Sections were stained with hematoxylin and eosin (HE, Merck) or used for enzyme- or immunohistochemistry (IHC).

For dewaxing and rehydration sections were passed through xylol (#9713.3, Roth, Karlsruhe, Germany) and decreasing concentrations of EtOH (#200-678-6; Fisher Scientific, Waltham, Massachusetts, USA) until the solution evenly flowed across the slide. Staining with Mayer´s hematoxylin solution consisting of 0.1% hematoxylin (#1.04302.0100, Merck, Darmstadt, Germany), 0.02% sodium iodate (#6525; Merck, Darmstadt, Germany), 5% potassium aluminum sulfate (#8896.1; Roth, Karlsruhe, Germany), 5% chloralhydrate (#K318.1; Roth, Karlsruhe, Germany) and 0.1% citric acid (#3958.1; Roth, Karlsruhe, Germany) for 1 min was followed by blueing in running tap water for 3 min. Slides were incubated in eosin solution consisting of 10% Eosin G (#7089.2, Roth, Karlsruhe, Germany) and 2 drops of glacial acetic acid (#3738.1; Roth, Karlsruhe, Germany) in 70% EtOH (#200-678-6, Fisher Scientific, Waltham, Massachusetts, USA) for 30 s and subsequently rinsed in ddH₂O before mounting.

Mammary gland whole mount analysis was carried out as described by Kleinberg et al. [23] (link). All chemicals came from Merck (Darmstadt, Germany), unless otherwise stated. After harvesting, tissues were fixed in ice-cold 4% paraformaldehyde for 2 h. Mammary glands were stored in 70% ethanol until further analysis. In short, following 3 washes with aceton for 30 min, mammary glands were rehydrated in 100% and 95% ethanol (30 min each). The glands were stained for 1 h with filtered hematoxylin, followed by thorough rinsing with tap water. Background staining was minimized by incubation (3×35 min) in a solution of 150 ml 100% ethanol, 150 ml aqua dest. and 7.5 ml HCl 1N (Sigma-Aldrich Chemie GmbH, Munich, Germany). Subsequently tissues were dehydrated in 70%, 95% and 100% ethanol (2×30 min for each step), followed by an incubation in Xylol (Roth, Karlsruhe, Germany) overnight. For long term storage mammary gland whole mounts were placed in methylsalicylate. Digital images of squash preparations were taken by a transmitted-light microscope stand mounted with a NIKON D200 camera (Nikon GmbH, Düsseldorf, Germany) at a distance of 60 cm all in one day. Picture analysis was performed using MetaVue software (MetaVue, Universal Imaging Corp., Downingtown, PA, USA) and ImageJ [34] (link). Pixel size was 11.125 µmx11.125 µm and pixel area was 123.766 µm².

At the end of the experiment, we excluded the data of one laying hen of the FP group. It showed a constant decrease in body weight until the end of the experiment. Thus, we analyzed seven laying hens kept in SC and six laying hens kept in FP.
The animals were weighed once a month from their 17th week of life until the end of the experiment at their 75th week of life.
Then, all animals were anesthetized with isoflurane and killed by bleeding. Both adrenal glands were extracted and fixed in 4% formaldehyde (Formalin, RotiHistofix, Roth, Karlsruhe, Germany). After 48 h, the adrenal glands were dehydrated with denatured ethanol and xylol (both Roth, Karlsruhe, Germany) and embedded in paraffin wax (Paraplast PLUS, Roth, Karlsruhe, Germany). The fixed organs were cooled at 4 °C until cutting. Subsequently, the adrenal glands were cut into 4 μm thin sections using a microtome (Leica, Wetzlar, Germany). Every 5th section was fixed onto a covered slide with a mixture of protein and glycerin (both Roth, Karlsruhe, Germany) and dried in an incubator at a temperature of 54 °C for an hour afterward. Afterward, the slices were stained with hematoxylin and eosin (Roth, Karlsruhe, Germany).