C apochromat water immersion objective na 1

Human ovarian cortical tissue was cut in 5 μm sections and mounted on glass slides. Dehydration and antigen retrieval was performed as described elsewhere (Stubbs et al., 2005 (link)) followed by serum block (30 min), then the primary antibody; (1/200) anti-TAFII68 Rabbit pAb (Bethyl Laboratories, #IHC-00094), (1/500) anti-FUS Rabbit pAb (Bethyl Laboratories, #A300-302A), or (1/200) anti-EWS Rabbit pAb (Bethyl Laboratories, #IHC-00086) overnight at 4°C. The sections were then incubated in a 1:700 dilution of secondary antibody (Donkey-anti-Rabbit) conjugated with Alexa Fluor 488 Dye (Life Technologies). Finally, sections were incubated in 1/7500 Hoechst (Life Technologies) followed by mounting with Dako Fluorescent Mounting Medium (Agilent Technologies, Santa Clara, CA, U.S.A.) and analyzed using a LSM510 laser-scanning confocal microscope using a 63x C-Apochromat water immersion objective NA 1.2 (Carl Zeiss, Göttingen, Germany) and ZEN 2011 software (Carl Zeiss, Göttingen, Germany).

Ovarian cortical tissue was sectioned in 5 μm slides and mounted on glass slides. Dehydration and antigen retrieval was performed as described elsewhere (Stubbs et al., 2005 (link)) followed by serum block (30 min), then primary antibody; anti-IGF2 rabbit polyclonal antibody (ab9574, Abcam, Cambridge, U.K.), (5 μg/ml) overnight at 4°C. This antibody was previously used and validated (Huang et al., 2010 (link)) and several other applications (ab9574-references.html">http://www.abcam.com/igf2-antibody-ab9574-references.html). The sections were subsequently incubated in a 1:250 dilution of appropriate secondary antibody (donkey-anti-rabbit for IGF2) conjugated with Alexa Fluor 488 Dye (Life Technologies, Carlsbad, CA, U.S.A.). Sections were incubated in 1/3,500 Hoechst (Life Technologies, Carlsbad, CA, U.S.A.) followed by mounting with Dako Fluorescent Mounting Medium (Agilent Technologies, Santa Clara, CA, U.S.A) and analyzed using a LSM510 laser-scanning confocal microscope using a 63x C-Apochromat water immersion objective NA 1.2 (Carl Zeiss, Göttingen, Germany). Zen 2011 software (Carl Zeiss, Göttingen, Germany) was used for analysis and image capturing. The quantification of IGF2 immunofluorescence was done by as ImageJ (Jensen, 2013 (link)).

Cryo sections (15 μm thickness) were blocked in 5% donkey serum PBS/Triton X-100 0.25% for 1 hour at RT. Primary antibodies (anti α₁ (1:400) (a6f-c, Developmental Studies Hybridoma Bank); anti α₃ (1:300) (06172, EMD Millipore, US); anti calbindin (1:400) (ab82812, Abcam, Cambridge, UK) were applied in 1% donkey serum PBS/Triton X-100 0.25% overnight at 4°C. Secondary labelling was done with Alexa Fluor fluorescent-conjugated secondary antibodies (Alexa Fluor 488 donkey anti rabbit (A21206, Life Technologies, Carlsbad, CA, USA); Alexa Fluor 568 donkey anti mouse (A10037, Life Technologies, Carlsbad, CA, USA) (1:350) in 1% donkey serum PBS/Triton X-100 0.25% for 1 hour at RT. Hoechst (1:10000) (Life technologies, Carlsbad, CA, USA) in PBS was used to counterstain the nuclei. Sections were mounted using fluorescence mounting medium (Dako, Glostrup, Denmark) and analysed on a LSM510 laser-scanning confocal microscope using a 40x C-Apochromat water immersion objective NA 1.2 (Carl Zeiss, Göttingen, Germany). Zen 2011 software (Carl Zeiss, Göttingen, Germany) was used for analysis and image capturing.