Palm laser dissection microscope

Serial 10-μm frozen sections were cut onto P.A.L.M. membrane slides (Zeiss) previously treated with UV exposure for 30–40 minutes. To delineate gland outline in frozen material, sections were subjected to dual enzyme histochemistry for cytochrome c oxidase and succinate dehydrogenase as per previously published protocols.²⁰ (link)^,22 (link) In all cases, sections were left to dry, and then microdissection was performed using a P.A.L.M laser dissection microscope (Zeiss). Microdissected cells were digested in 14 μL Picopure digestion buffer (Life Technologies) at 65°C for 3 hours, followed by proteinase K inactivation at 95°C for 5 minutes.

To determine AMH gene expression in granulosa (luteal) cells and theca (luteal) cells, first immunostaining was performed on methacarn fixed tissue according to the methods described above to identify the AMH-positive cells. The subsequent sections were stained with Mayer’s haematoxylin and used to perform laser capture micro-dissection (LCM), according to DeCarlo et al. [37 ] with minor modifications. Briefly, after staining, sections were dehydrated and air-dried for 5 minutes. AMH positive granulosa cells of an antral follicle from an ovary in the early folicular phase and theca cells of a pre-ovulatory follicle from an ovary in the late follicular phase as well as corpora luteal cells from the ovary in the luteal phase of the oestrus cycle were micro-dissected under 40x magnification (Palm Laser Dissection Microscope, Zeiss, Sliedrecht, the Netherlands). Around 1x10⁴ granulosa, theca or corpora lutea cells were collected into silicon coated Adhesive cap500 caps (Zeiss, Gottingen, Germany), respectively and analysed separately. After microdissection, the cells in the caps were lysed with 20 μl of extraction buffer (Picopure RNA Isolation kit, Arcturus, San Diego, CA, USA) and incubated for 30 min at 42°C. The resulting cell lysates were stored at -80°C until RNA isolation.