X71 microscope

The 135 pairs of resected HCC and para-cancerous tissues were fixed overnight with 4% paraformaldehyde, embedded in paraffin and cut into 4 mm thick sections. Immunohistochemical staining was performed with an Envision IHC kit (Maxin Biotechnologies Inc., Fuzhou, Fujian, China). Briefly, the tissue sections were dewaxed and hydrated, followed by antigen retrieval with citrate buffer for 15 min at 100 °C in a microwave oven. The sections were incubated with the primary antibody against HSF1 (Santa Cruz Biotechnology, Inc., USA) at room temperature for 1 h at a dilution of 1:200 and were visualized using the UltraVision Quanto Detection System HRP DAB kit (Thermo Scientific) according to the manufacturer’s protocols. The stained sections were counterstained with haematoxylin and then developed according to the manufacturer’s instructions and scored using an Olympus X71 microscope. Based on the staining intensity, samples were divided into the following grades: 0: < 10% positive staining HCC cells; 1+: 11–25% positive staining HCC cells; 2+: 26–50% positive staining HCC cells; 3+: > 50% positive staining HCC cells. IHC and scoring analysis were performed independently by two investigators.

Cells were seeded at a density of 2500/cm² in 12 well-plates on coverslips and cultured under standard conditions. After two days, cells were treated with peptides for another 24 h. Cells were than fixed with 4% PFA and stained with phalloidin (1:200, Molecular Probes, Life Technologies) overnight and Hoechst (1:10000) for additional 5 min. Coverslips were mounted on slides with Immu-Mount (Thermo scientific, Massachusetts, USA). Pictures were taken with Olympus X71 microscope with x1000 magnitude. Exposure time was equal in different cell lines. Images were taken with cell- F software (Olympus, Tokyo, Japan). Cell length and nuclear morphology was measured by image J software (NIH, USA).

Following tryspinization for 5 minutes, cell lines were suspended in PBS and spun down on a microscope slide (10,000/spot), then the fixed with 2% formaldehyde in PBS. The staining procedure was performed at room temperature using the same protocol for tissues and cell lines, as follows. Endogenous peroxidase blockade was performed in 0.3% V/V H₂O₂ for 5 minutes. Following permeabilization with 0.15% V/V Triton-X100 in PBS, samples were rinsed in PBS and blocked with blocking buffer (5% FBS + 0.05% Tween-20 in PBS) for 1 hour, then incubated with the primary antibody (10 μg/mL in blocking buffer) for 1 hour. After 3 washing steps in blocking buffer (5 minutes each), samples were incubated with the corresponding secondary antibody (2 μg/mL in blocking buffer) for 1 hour. HRP-DAB reaction was carried out for 5 minutes after 3 additional washes, and stopped in water. Slides were mounted with a glycerol-based medium before taking pictures with an inverted Olympus X71 microscope. Antibodies were as follows: rabbit anti-SP17 and mouse anti-PTTG1 (Abcam), goat anti-AKAP4 (Santa Cruz). All secondary antibodies (HRP-linked) were from Abcam.

Cell proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide (MTT) assay as describe before20 (link). Cell death was monitored by propidium iodide (PI) staining. Briefly, cells were seeded at 3000 cells/cm² in 96-well plate and incubated at standard conditions. After 12 h, the cells were treated with scorpion peptides. Forty-eight hours after treatment, cells were incubated with MTT solution (Roth, Karlsruhe, Germany) (5 mg/ml) or PI (Sigma, Germany) (3 μg/ml), at 37 °C, 5% CO₂. After 4 h MTT incubation or 10 min of PI incubation, images were obtained using with Olympus X71 microscope (Olympus, Hamburg, Germany) with a long-distance 10 × objective. Exposure time was equal in different groups. Images were taken with cell- F software (Olympus). Cells with 4 h MTT incubation were then lysed with 100 μl acidic isopropanol. OD values were determined with a SLT spectra devise (Crailsheim, Germany) using Tecan × Fluor4 software for quantification.