Vectashield vibrance antifade mounting medium with dapi

Propidium iodide was used in place of IR780 to evaluate cell internalization prior to dye release. Propidium iodide usually emits green fluorescence, but when bound to nucleic acid, red fluorescence is instead emitted. Propidium iodide is usually impermeable to the cell membrane, but can be allowed entry into the cell by encapsulation within TROS. S2VP10 cells were grown to confluency in a 6-well plate containing 12mm cover glasses in wells. Cells were grown overnight in DMEM with 10% FBS and 1% L-glutamine. pH-specific DMEM (pH 7.4 or 6.6) was used to mimic benign and malignant conditions for pH-specific internalization and release. 80 μL of PBS (control) or V7-TROS with propidium iodide was added to the plate for 1.5 hr. Plates with then fixed with 4% formaldehyde for 10 min at room temperature. Cells were washed with PBS to remove free TROS and propidium iodide. The cover glasses were removed and mounted with VectaShield Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA). The samples were dried in the darkness for 2 hrs. Images were taken at 400X magnification using a Leica SP8 Confocal Microscope. Fluorescence counts of images were determined through channel splitting and thresholding via ImageJ.

Tissues were fixed overnight in 10% formalin, dehydrated in ethanol, embedded in paraffin, and cut into 5 µm sections. Picrosirius Red staining was performed with the Picro Sirius Red Stain Kit (Abcam) according to the manufacturer’s instructions. Masson’s trichrome staining was performed with the Masson’s Trichrome Stain Kit (Polysciences) according to the manufacturer’s instructions. For immunofluorescence staining, sections were de-paraffinized with Histo-Clear II (National Diagnostics) and rehydrated according to the manufacturer’s instructions. Antigen retrieval was performed for 40 min in citrate buffer pH 6.0 (Vector Laboratories) in a steamer (IHC World). Sections were blocked in 5% BSA and 5% normal goat serum (Cell Signaling) in TBS containing 0.1% Tween-20, and incubated in primary antibodies at 4°C in a humidified chamber overnight. Sections were incubated in secondary antibody in blocking solution for 1h at room temperature and mounted in Vectashield Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories). The following primary antibodies were used: SMA (1:400; Millipore, CBL171), CK8 (1:200; DSHB, TROMA-I). The following secondary antibodies were used: donkey anti-mouse Alexa-Fluor 488, donkey anti-rat Alexa Fluor 647 (1:1,000; Thermo Scientific).