Three-Dimensional Imaging of Adult Ovary

Adult ovaries were processed according to standard procedures (35 (link), 62 (link), 63 (link)). Images were acquired using a Zeiss LSM710 Confocal microscope and the Leica laser scanning confocal microscope TCS SP8, deconvolved using Huygens Software (Scientific Volume Imaging B.V.), projected using Fiji Software, and treated with Adobe Photoshop 2017 (Adobe Microsystems). Three-dimensional projections were generated with LAXs software from immunofluorescence images acquired using the Leica laser scanning confocal microscope TCS SP8. In Coherent-hybrid STED (CH-STED) Superresolution Imaging, 90% glycerol, 0.5% N-propyl gallate, and 20 mM Tris⋅HCl (pH 8) were used as mounting medium. The superresolution CH-STED images were acquired using Abberior Instruments “Expert Line” gated-STED coupled to a Nikon Ti microscope, an oil-immersion 60× 1.4 numerical aperture Plan-Apo objective (Nikon, Lambda Series), and a pinhole size of 0.8. The coherent-hybrid mode was previously described (66 (link)) and is set by imprinting a bivortex phase map (ρ = 0.92) onto a spatial light modulator (Hamamatsu LSH 0801392). Used antibodies are shown in SI Appendix, Table 1F.

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Feijão T., Marques B., Silva R.D., Carvalho C., Sobral D., Matos R., Tan T., Pereira A., Morais-de-Sá E., Maiato H., DeLuca S.Z, & Martinho R.G. (2022). Polycomb group (PcG) proteins prevent the assembly of abnormal synaptonemal complex structures during meiosis. Proceedings of the National Academy of Sciences of the United States of America, 119(42), e2204701119.

Publication 2022

LSM710 Confocal microscope Huygens Software Leica laser scanning confocal microscope TCS SP8 “Expert Line” gated-STED Plan-Apo objective

Adult Antibodies Confocal microscope Glycerol Hybrid Immersion Immunofluorescence Laser scanning confocal microscope Light Microscope Ovaries Propyl gallate Tris

Corresponding Organization :

Other organizations : i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, University of Aveiro, Algarve Biomedical Center, University of Algarve, University of Lisbon, Universidade Nova de Lisboa, Brandeis University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

None explicitly mentioned

control variables

Glycerol (90%)
N-propyl gallate (0.5%)
Tris·HCl (20 mM, pH 8) as mounting medium
Pinhole size (0.8) for Coherent-hybrid STED (CH-STED) Superresolution Imaging

controls

Positive control: None mentioned
Negative control: None mentioned

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