Alexa fluor 555 labeled secondary antibody

HCC cells were seeded in glass bottom dishes. After cells attached to the bottom of the dishes, cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. Afterwards, the cells were incubated with blocking buffer (5% BSA in PBS, pH 7.4) for 30 min. Cells were incubated with the appropriate primary antibodies overnight at 4 °C. On the following day cells were incubated with Alexa Fluor 555-labeled secondary antibodies (Beyotime, Haimen, China) for 1 h and DAPI was used to mark nuclei. For the TUNEL assays, after cells were incubated with blocking buffer, the TUNEL reaction mixture (Roche, Pleasanton, CA, USA) was used to treat cells for 1 h at 37 °C in the dark place. Subsequently, cells were strained with DAPI for nuclei visualization. Apoptotic cells appeared as red-stained cells. The cells were observed and photographed under a fluorescence microscope (Olympus, Tokyo, Japan). Data are shown from a typical experiment performed in triplicate.

Cells were fixed with a 4% paraformaldehyde solution for 15 min and washed for 5 min three times with PBS. The cells were then permeabilized and blocked with 0.2 % Triton X-100/PBS for 3 min, and then rinsed by PBS for 5 min three times. They were then blocked using 1% BSA/PBS for 1 h. Cells were incubated with primary antibodies anti-tenomodulin antibodies (dilution, 1: 200; Abcam, Cambridge, MA, USA) overnight at 4 °C, followed by Alexa Fluor 555-labeled secondary antibodies (dilution,1:500; Beyotime, Shanghai, China). Actin filaments and DNA were stained with Phalloidin-Tetramethylrhodamine Conjugate (Sigma-Aldrich, Darmstadt, Germany), and 500 nM DAPI (Beyotime, Shanghai, China), respectively. Images were captured with a Leica fluorescence microscope (Leica, Solms, Germany).

Cells were transplanted into 6-well plates covered with circular coverslips in the bottom and allowed to attach overnight. After different treatment of PTUPB for 48 h, cells were fixed by 4% paraformaldehyde. For phalloidin staining of actin fibers, fixed cells were incubated with fluorescein isothiocyanate (FITC)-labeled phalloidin (Beyotime Institute of Biotechnology, Shanghai, China) for 60 min at room temperature and then mounted onto slides for observation after nuclei staining. For HMMR staining, fixed cells were probed with HMMR primary antibody (OriGene, Rockville, MD, USA) at 4°C overnight followed by incubation with Alexa Fluor 555-labeled secondary antibody (Beyotime Institute of Biotechnology, Shanghai, China) for 60 min at room temperature. Hoechst 33258 was used for staining nuclei. Cell samples were observed and pictures were taken by using a Zeiss Axio Scope.A1 microscope (Carl Zeiss, Oberkochen, Germany).