Carl axio imager m1 microscope

Testes for light microscopy histopathologic analysis were weighed, fixed in Bouin’s solution for 24 h, rinsed, and dehydrated in alcohol. Tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. For immunofluorescence experiments, fresh testes were embedded in OCT compound (Sakura Finetek, Torrance, CA) and stored at −80°C before the preparation of 3μm sections, which were fixed for 30 min in PBS-buffered 4% paraformaldehyde at room temperature, followed by overnight in PBS-buffered 1% paraformaldehyde at 4°C. Sections were labeled with a primary antibody against CTNNB1 (8480, Cell Signaling, Danvers, MA) in PBS/0.3% Triton X-100 overnight at 4°C and subsequently probed with secondary Alexa Fluor 594 goat anti-rabbit (Molecular Probes, Eugene, OR) for 1 h at room temperature. Slides were mounted using VectaShield with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Labs). Photomicrographs were taken using a Carl Zeiss Axio Imager M1 microscope (Carl Zeiss Canada Ltd, Toronto, Canada) using Zen 2012 blue edition software (Carl Zeiss, Oberkochen, Germany).

For immunohistochemistry (IHC) analyses, bovine ovaries and sample selection for experimental or control groups were performed as described above. At the laboratory, entire ovaries (at least four per group) were fixed in 10% formaldehyde solution for 24 h, rinsed and dehydrated in alcohol until embedded in paraffin. Serial sections were prepared (at a thickness of 3 µm) followed by deparaffinization, rehydration, sodium citrate heat-mediated antigen retrieval, peroxidase block and protein blocking (10% goat for 30 min), and then slides were probed with primary antibody against total and phosphorylated forms of YAP or against Pan-TEAD (Table 1) overnight at 4 °C. Protein detection was then performed with the Vectastain Elite ABC HRP Kit (VECTPK6101; Vector Laboratories Inc., Burlingame, CA, United States) and stained with DAB substrate kit (VECTSK4100; Vector Laboratories Inc., Burlingame, CA, United States). Slides were then counterstained with hematoxylin and dehydrated with graded alcohols prior to mounting. Negative controls consisted of slides for which the primary antibody was omitted. Photomicrographs were taken using a Carl Zeiss Axio Imager M1 microscope (Carl Zeiss Canada Ltd., Toronto, ON, Canada) at ×1000 magnification and using the Zen 2012 blue edition software (Carl Zeiss, Oberkochen, Germany).