Meta laser scanning microscope

A microarray containing tissue from 40 OS patients was obtained from Alena Biotechnology Co., Ltd. (Xi’an, China). OS tissue sections were hybridized with the lncRNA KCNQ1OT1 (BioTNT Biotechnologies, Shanghai, China) and miR-34c-5p probes (Servicebio, Wuhan, China). Probe mix was denatured at 85 °C and hybridization was conducted at 65 °C overnight. Sections were washed using reducing concentrations of saline sodium citrate. Then slides were treated with 5% blocking solution for 30 min at room temperature. Each section was incubated with 100 μl HRP-labeled anti-DIG antibody at 1:500 in blocking buffer overnight at 4 °C. Then washed with TBS and TSA staining solution was created with a Perkin-Elmer TSA Plus kit according to the manufacturer’s instructions. Incubated in DAPI-containing TBS, then rinsed in water, air dried, and mounted in an aqueous fluorescence mounting media. Confocal microscopy (LSM 510, META laser scanning microscope, Zeiss) was used to acquire the images. The intensities of KCNQ1OT1 and miR-34c-5p staining were scored using the following staining criteria: 0–5% was scored as 0; 6–35% was scored as 1; 36–70% was scored as 2; and >70% was scored as 3. A total score <2 was considered to represent negative expression, and a score ≥2 was defined as a positive expression. The scoring was performed blind and determined by two senior pathologists.

Second harmonic generation (SHG) microscopy was performed on each ONH section using a META laser scanning microscope (Zeiss Ltd, UK), equipped with a 20× Plan-apochromat objective lens and multiphoton Ti:sapphire laser (Chameleon, Coherent UK Ltd, UK). Following excitation at 800 nm, the forward scattered signals from each ONH section were acquired as sequences of optical sections (256 × 256 × 1 pixel) at 1 µm increments of focus using an automated motorized stage. These were tiled together using LSM 510 v. 4.2 SP1 (Carl Zeiss, UK) to create three-dimensional image stacks of each ONH section. In this study, the latter was solely used to visualize the collagenous architecture of the ONH throughout each 100 µm tissue section so as to determine which sections contained prelamina, LC and/or postlaminar tissue. These three-dimensional image stacks were then converted to maximum intensity projections (MIPs) using ImageJ (1.45) software to ensure confident differentiation of each ONH region of interest, so that data selection and analysis for each region could be performed subsequently.

Cells were seeded at 12-well U-Chamber (Ibidi, Germany), fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.05% Triton X-100 for 1 min at room temperature. Primary antibodies used in immunofluorescence staining were vinculin (EPR8185; Epitomics) and paxillin (ab32084; Abcam). The nucleus was stained with DAPI (Sigma-Aldrich, USA). Images were acquired by confocal microscopy (LSM 510, METALaser Scanning Microscope, Zeiss). The raw density was assessed using ImageJ.

A BeyoClick™ EdU Kit (C0075S, Beyotime Biotechnology, Shanghai, China) was used to perform the EdU assay following the manufacturer's guides. Cells were plated in a 12-well chamber (Ibidi, Germany) and supplied with 200 μl EdU (50 μM) for 2 h incubation. Subsequently, cells were added with 4% paraformaldehyde for 15 min and permeabilized with 0.3% Triton X-100 for 10 min and then labeled with 5 μg/ml of Hoechst 33342 for 30 min. The images were obtained by a confocal microscope (LSM 510, META laser scanning microscope, Zeiss).

In cell assay, all the HCC cell lines under tests were seeded on cover slides in 24-well plates and incubated overnight. For F-actin staining, cells were incubated with phalloidin-FITC (Sigma-Aldrich) for 75 minutes at room temperature. For EDIL3 staining, cells were incubated with primary antibodies against EDIL3 (Abnova) for 75 minutes at room temperature, followed by an Alexa Fluor 594-conjugated secondary antibody. In tissue staining, two samples embedded in paraffin were subjected to heat-mediated antigen retrieval in pH 6.0 citric acid, and blocked by 10% BSA. The primary antibodies used are EDIL3 (Abnova) and CD31 (Abcam) with Alexa Fluor 594-conjugated secondary antibody against EDIL3 and Fluor 488 against CD31. Immunofluorescence signals were captured using confocal micro-scopy (LSM 510, METALaser Scanning Microscope, Zeiss).