Instruments confocal microscope

HCC cells were fixed with 4% paraformaldehyde and permeabilized using 0.3% Triton X-100 for 15 min. Then, the fixed cells were incubated with anti-CD44 primary antibody overnight. The secondary antibody was an Alexa Fluor-conjugated IgG (Invitrogen, Carlsbad, CA, USA). The cells on the slides were imaged and visualised with the appropriate excitation and emission spectra at a magnification of ×400 using a Zeiss Instruments confocal microscope (Zeiss, Oberkochen, Germany).

After treatment with LL-37 (0, 8, 16 µM) for 24 h, PANC1 and MIA PaCa-2 cells were washed with phosphate-buffered saline and fixed in 4% paraformaldehyde. After permeabilization with 0.5% Triton X-100 and blocking in 5% bovine serum albumin (BSA), cells were incubated with primary antibodies specific for LC3B and γ-H2AX overnight at 4°C, followed by fluorescence-conjugated secondary antibodies at room temperature for 1 h. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. Pseudocolor images were obtained using a Zeiss Instruments confocal microscope (Carl Zeiss, Oberkochen, Germany).

Cells were fixed in 4% formaldehyde diluted in phosphate-buffered saline (PBS) for 15 min, permeabilized with 0.3% Triton X-100, treated with blocking buffer (5% BSA in PBS), and then incubated overnight with the primary antibody at 4°C. The cells were then incubated with the Red-conjugated secondary antibody from Jackson ImmunoResearch Laboratories (West Grove, PA, USA) for 1 h at room temperature. Slides were mounted and examined using a Zeiss Instruments confocal microscope.

HSCs cultured on polyacrylamide hydrogels with different stiffness (0.4 kPa or 25.6 kPa) or human liver cryosections were prepared for IF staining, as previously described5 (link), 13 (link). Briefly, HSCs or human liver cryosections were fixed with 4% PFA (paraformaldehyde) for 15 minutes followed by permeabilization by 0.3% Triton X-100 for 5 minutes. After washing the cryosections with 1 x PBS three times, 5% BSA was administrated to block non-specific antibody binding sites. The cryosections were then placed in primary antibody diluted in blocking buffer and incubated overnight at 4°C. After rinsing with 1 x PBS three times, the samples were then incubated with fluorescent secondary antibody, and DAPI respectively. The samples on the slides were visualized with a Zeiss Instruments confocal microscope (Cal Zeiss AG, Jena, Germany). ImageJ was utilized to quantify the immunofluorescence density of each image.

8505C cells were treated with 5 ng/mL TGF-β1 for 48 h. Morphological changes in the 8505C cells were observed under a Leica DMIL LED phase contrast microscope with an attached EC3 camera (Leica, Germany). The photographs were taken at 200 x magnification. Immunofluorescence staining was used to detect E-cadherin, cytokeratin, vimentin, and F-actin in the 8505C cells as the manufacture’s instruction. The fluorescent images were obtained by confocal laser scanning microscope (Zeiss LSM 510, Oberkochen, Germany).
After 8505 cells were maintained in media supplemented with 5 ng/mL TGF-β1 with or without 60 μM EGCG for 48 h. 8505C cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde. After
permeabilization with 0.5% Triton X-100 and blocking in 5% bovine serum albumin (BSA), the cells were incubated
with primary E-cadherin or vimentin antibodies overnight at 4°C then incubated with fluorescence-conjugated secondary antibodies at room temperature for 1 h. The nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI) for 5 min. Images were pseudocolored using a Zeiss Instruments confocal microscope.

Ad-mRFP-GFP-LC3 was purchased from HanBio Technology Co. Ltd. (HanBio, Shanghai, China). PC cells were transfected as previously described [32 ] and then cultured with different concentrations of glucose for 6 h. Images were taken with a Zeiss Instruments confocal microscope at 400× magnification.