Ultrastructural Analysis of Granulosa Cells

After fixation, the tissue samples were rinsed in phosphate-buffered saline solution and postfixed in 1% osmium tetroxide, dehydrated through an ascending series of ethanol, immersed in propylene oxide, and embedded in Epon 812 (Agar Scientific, Essex, UK). Afterwards, ultrathin (60–90 nm) sections were obtained using a Leica EM UC7 ultramicrotome (Wetzlar, Germany) mounted on copper grids, contrasted with saturated uranyl acetate and lead citrate, and examined by transmission electron microscopy (TEM, Zeiss-EM 109 electron microscope, Germany). The appearance, organelles distribution, membranes integrity, and connections between the granulosa cells and the oocyte were investigated.

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Moghassemi S., Dadashzadeh A., Camboni A., Feron O., Azevedo R.B, & Amorim C.A. (2023). Ex vivo purging of cancer cells from ovarian tissue using photodynamic therapy: a novel strategy to restore fertility in leukemia patients. Human Reproduction Open, 2023(2), hoad005.

Publication 2023

Epon 812 EM UC7 ultramicrotome EM 109 electron microscope

Agar Citrate Copper Dehydrated ethanol Electron microscope Epon 812 Granulosa cells Membranes Oocyte Organelles Osmium tetroxide Phosphate Propylene oxide Saline solution Tissue Transmission electron microscopy Ultramicrotome Uranyl acetate

Corresponding Organization : UCLouvain

Other organizations : Cliniques Universitaires Saint-Luc, Universidade de Brasília

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Variable analysis

independent variables

Fixation protocol (not explicitly mentioned)
Osmium tetroxide concentration (1%)
Dehydration procedure (ascending series of ethanol)
Embedding medium (Epon 812)

dependent variables

Appearance of granulosa cells and oocyte
Organelles distribution
Membranes integrity
Connections between granulosa cells and oocyte

control variables

Phosphate-buffered saline solution (for rinsing)
Propylene oxide (for immersion)
Ultrathin section thickness (60-90 nm)
Copper grids (for mounting sections)
Uranyl acetate and lead citrate (for contrasting)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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