The spheroids were fixed for 2 hours at room temperature in 4% paraformaldehyde and subsequently dehydrated in 30% sucrose for at least 2 days at 4°C. The spheroids were embedded and frozen using Tissue-Tek OCT (Sakura Finetek, Alphen aan den Rijn, The Netherlands) and sectioned at 8 μm using a NX70 Cryostat (Thermo Fisher). Spheroid sections were blocked using 5% BSA and 0.25% Triton X-100 in PBS (PBT, Sigma-Aldrich) for 2 hours at room temperature. A list of the antibodies is provided in Supplemental Table S2, http://links.lww.com/HC9/A782 .
The spheroid sections were stained with a primary antibody diluted in PBT overnight at 4°C. The slides were washed with PBS 3 times. Next, the secondary antibody diluted in PBT was added and incubated for 2 hours at room temperature in the dark. The slides were then washed 3 times with PBS. The slides were mounted with ProLong Gold Antifade containing DAPI (Thermo Fisher) and imaged using an Olympus IX73 inverted microscope (Olympus, Tokyo, Japan). Images were analyzed using the Fiji software version 2.12.0. For quantification, the integrated density of the respective staining was normalized to the area stained with DAPI. Every data point represents the average fluorescence intensity of at least 10 imaged spheroids.
NileRed (Sigma-Aldrich) staining was performed as described.13 (link)
The spheroid sections were stained with a primary antibody diluted in PBT overnight at 4°C. The slides were washed with PBS 3 times. Next, the secondary antibody diluted in PBT was added and incubated for 2 hours at room temperature in the dark. The slides were then washed 3 times with PBS. The slides were mounted with ProLong Gold Antifade containing DAPI (Thermo Fisher) and imaged using an Olympus IX73 inverted microscope (Olympus, Tokyo, Japan). Images were analyzed using the Fiji software version 2.12.0. For quantification, the integrated density of the respective staining was normalized to the area stained with DAPI. Every data point represents the average fluorescence intensity of at least 10 imaged spheroids.
NileRed (Sigma-Aldrich) staining was performed as described.13 (link)
