Cellsens dimensions imaging software

To determine the expression of reporter genes after CRISPR/Cas13b activity along with guide RNA targeting, transfected HEK293T cells were fixed in 3.7% formaldehyde for 10 min at room temperature and then permeabilized with 0.2% Triton X-100 in PBS for 10 min. Subsequently, Fluoroshield mounting medium with DAPI (Abcam, USA) was dropped onto the cells, and imaging was performed by a Olympus IX73 inverted microscope with cellSens Dimensions imaging software (Olympus, Japan).

A piece of lung or liver freshly isolated from adult mice or embryos at E18.5 was immediately frozen in OCT compound for cryostat sectioning (5 μm per section). The frozen tissue sections were fixed with acetone for 15 min at −20 °C. After rinsing with PBS for 5 min, samples were blocked with anti-FcγRII/III antibody at RT for 15 min followed by staining at 4 °C overnight with PE-labeled anti-Siglec-F (adult lung) or PE-labeled anti-F4/80 (fetal lung) antibody. The samples were then stained with 1 μg/ml DAPI at RT for 1 min and mounted under a coverslip with one drop of IMMU-MOUNT (Thermo Fisher Scientific). Specimens were visualized on an Olympus FSX100 fluorescence microscope (Olympus Scientific Solutions Americas Corp, Waltham, MA) at ×10 magnification. At least 5 independent images were collected from each specimen. Images were collected using FSX-BSW (version 03.02.12) software (Olympus). The cell density of AMs (Siglec-F⁺) or preAMs/TRMs (F4/80⁺) was measured based on cell numbers counted in the fields using cellSens Dimensions Imaging Software version 1.15 (Olympus).

Immunolabelling was visualized using a BX51 epifluorescence microscope (Olympus, Parkside, SA, Australia) equipped with narrow filters for Alexa Fluor^® 488 and 568. An XM10 monochrome camera (Olympus, Parkside, SA, Australia) was used to acquire the images. CellSens Dimensions Imaging Software (Olympus, Parkside, SA, Australia) was used to adjust the brightness and contrast.
Cell counts were performed in 5–6 non-consecutive sections per gastric region and animal. Immunopositive cells were manually counted in a 159 × 159-µm square area at the base of the glandular region (i.e., location of gastric ghrelin [39 (link)] and CaSR [40 (link)] immunopositive cells) using the software FIJI [41 (link)].