Tcs8 confocal microscope

Slice cultures were fixed for 20 minutes in 4% paraformaldehyde in PBS. Slices were washed twice in PBS, blocked for 1 hour in blocking solution (PBS with 0.5% Triton X-100% and 3% goat serum) then incubated in 200 μL primary antibody diluted in blocking solution overnight at 4 °C with shaking. Slices were washed 3 times in PBS before being incubated (2 hours, RT in the dark) with secondary antibodies in the blocking solution. In the second of the final 3 PBS washes, slices were incubated with DAPI (Sigma, 1:10,000 in PBS). Images were captured using a Leica TCS8 confocal microscope (Leica, Wetzlar, Germany). Primary antibodies used: rat Lamb1 (1:250; Abcam #ab44941), rabbit Calnexin (1:250; Abcam #ab13504). Secondary antibodies used: goat anti-rabbit Alexa Fluor 594 (1:400; Invitrogen #A11037) and goat anti-rat Alexa Fluor 647 (1:400; Invitrogen #A21247).

The efficiency of siFAP was assessed by measuring the expression levels of FAP using flow cytometry. LX-2 cells were cultured in DMEM supplemented with 10% FBS, 100 units/mL of penicillin, and 100 μg/mL of streptomycin. LX-2 cells (4 × 10⁵ cells/well) were seeded to a six-well plate (SPL Life Sciences), incubated for 24 h, and transfected for 20 min with 50 nM siFAP (Bioneer Corporation, Daejeon, Republic of Korea) complexed with 5 μL of Lipofectamine 2000. The sequences of siFAP were 5′-CUC UAU GCA GUG UAU CGA AdTdT-3′ (sense) and 5′-UUC GAU ACA CUG CAU AGA gdTdT-3′ (antisense). In some experiments, scrambled-sequence siRNA (siSCR) was used as a control. After transfection, cells were incubated for an additional 48 h. Next, cells were stained with a rabbit anti-FAP primary IgG antibody (1:50, Abcam) for 1 h, followed by an allophycocyanin (APC)-conjugated goat anti-rabbit IgG antibody (1:100, Abcam). After staining, cells were analyzed via BD LSR Fortessa (BD Bioscience) and Leica TCS8 confocal microscope (Leica, Bensheim, Germany). Flow cytometry data were acquired with BD FACSDIVA™ (v8.0.1., BD Bioscience). LEICA Application Suite X (v3.6.0., Leica) was used to collect confocal images. Control cells were stained with rabbit IgG isotype antibody (1:50, Abcam) followed by an APC-conjugated goat anti-rabbit IgG antibody (1:100, Abcam).