Sm200r sliding microtome

After washing in PBS, the dissected mammary glands and tumors were fixed in 4% paraformaldehyde–PBS overnight and for 2 days, respectively. After washing in PBS, samples were dehydrated gradually from 70% ethanol to 100% ethanol and then from 50% xylene in ethanol to 100% xylene, following paraffin replacement using a Leica ASP300 fully automatic closed tissue processor and paraffin-embedding using a Leica EG1160. The paraffin-embedded tissues were then cut into 5-μm thick sections using a Leica SM200R sliding microtome. The sections were gradually deparaffinized in xylene and then with ethanol, gradually decreasing from 100% to 50% ethanol, and washed with distilled H₂O, and then stained in hematoxylin solution (0.25% hematoxylin (Nacalai Tesque), 0.05% sodium iodate (Nacalai Tesque), 12.5% potassium alum (Wako), and 0.25% citric acid (Wako)) for 10 min. After washing in distilled H₂O, sections were blued in 0.1% saturated lithium carbonate at 37 °C for 5 min and then washed in distilled H₂O and stained in eosin solution (1% eosin (Wako) and 0.02% glacial acetic acid) for 10 min. After washing in 90% and 100% ethanol, sections were soaked in xylene for 5 min and then mounted in MGK-S (Matsunami).

Pups were perfused transcardially with 0.9% saline. Whole brains were dissected and immersed in the Golgi–Cox solution (5% potassium dichromate, 5% mercuric chloride, and 5% potassium chromate) for ~2–3 weeks. Then, brains were transferred to a 30% sucrose solution and stored in the dark at 4 °C. Coronal slices (100 µm-thick) were cut in 6% sucrose with a microtome (Leica SM200R sliding microtome) and transferred onto 2% gelatin-coated slides to initiate the staining process in humidified chambers. Ammonium hydroxide was applied for 30 min. Slices were then treated for 30 min with 1% sodium thiosulphate. Next, slices were treated with an increasing grade of ethanol starting from 50 to 100%, followed by a 15-minute immersion in solution X (1/3 parts by volume of chloroform, 1/3 parts by volume of xylene and 1/3 parts by volume of 100% ethanol). Slices were then treated with xylene for 15 min, mounted in mowiol, and examined under bright field with an upright Olympus BX51W microscope using a ×63 oil-immersion objective (N.A. 1.4). Spine density was calculated as for DiI staining.