Ds qi2 microscope

Frozen mouse conceptuses were embedded in O.C.T. compound, cryosectioned at 10 μm, and fixed in 4% paraformaldehyde. Sections were then treated with 0.3% hydrogen peroxide in methanol to block endogenous peroxidases, permeabilized using 0.3% Triton-X, and blocked with 10% normal goat serum (ThermoFisher Scientific). Sections were immersed in a primary antibody specific for cytokeratin (628602, 1:400, BioLegend, San Diego, CA, USA), and then incubated with a mouse biotinylated secondary antibody, followed by Vectastain peroxidase (Vector Laboratories, Burlingame, CA, USA). Placental sections were then treated with an AEC Chromogen Solution (ThermoFisher Scientific), nuclei counterstained with hematoxylin, and sections mounted using Fluoromount-G (Southern Biotech, Birmingham, AL, USA). For fluorescent staining, sections were fixed and permeabilized as described above, and incubated with antibodies specific for TPBPA (104401, 1:100, Abcam, Toronto, ON, Canada), or cytokeratin conjugated to Alexa 488 (628608, 1:400, BioLegend). Sections immersed in antibodies specific for TPBPA were washed and then incubated with Alexa 555-conjugated anti-rabbit secondary antibodies. All sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, ThermoFisher Scientific), mounted, and imaged using a Nikon DS-Qi2 microscope.

In situ hybridization was conducted using RNAScope, as per the manufacturer’s instructions (Advanced Cell Diagnostics, Newark, CA, USA). Briefly, sections were hydrated through a graded series of ethanol washes, subjected to peroxidase and protease treatment, and hybridized with probes specific to mouse Ovol2. Additional sections treated with probes specific for mouse Ppib and bacterial dapB served as positive and negative controls, respectively. Sections were then subjected to a series of amplification steps, treated with 3,3′-diaminobenzidine chromogen solution, and nuclei were counterstained with hematoxylin. Sections were dehydrated using increasing concentrations of ethanol, cleared using xylene, mounted with Cytoseal (ThermoFisher Scientific), and imaged using a Nikon DS-Qi2 microscope.

Embryos were collected and processed for histology, immunostaining, or in situ hybridization as described previously (Xu et al., 2016 (link)). For histology and immunofluorescent staining, the embryos were fixed in 4% paraformaldehyde (PFA), dehydrated through an ethanol series, embedded in paraffin, and sectioned at 7 μm thickness. The goat anti-Foxf1 (AF4798; R&D) antibody was used to detect the Foxf1 protein. Images were taken using a Nikon DS-Qi2 microscope (Nikon Instruments Inc., Melville, NY, United States).