Imagescanner 3

The images of the total protein pattern of 2D-gels were captured by video imaging using the ImageScanner III (LabScan 6.0 software, GE Healthcare, Life Sciences, Sweden). We then used a specialized software, Progenesis Samespots, version 3.1 (Non-linear Dynamics, Newcastle upon Tyne, UK) to align and quantify the protein spots from captured 2D-gel images. The use of identical spot boundaries across all gels, background subtraction, and normalization to total staining intensity in each gel ensured comparable data between all gels with the Progenesis Samespots software. Each spot was manually evaluated using a 3D image display to exclude artifacts (spiky or irregularly shaped spots, split spots etc.). For the identified real spots, image realigning, noise filtering, and spots segmentation were carried out using default setting [32 (link)]. Automatic analysis was performed on all the aligned images using the analysis wizard. The aligned images were grouped into high and low n-3 PUFA group, and the statistically ranked list of spots was evaluated.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out as previously described [38 (link)]. The sample was mixed with an equal volume of protein loading buffer (P1016, Solarbio, Beijing, China) and centrifuged at 10,000 r/min for 5 min. Then, 10 μL of the supernatant were loaded into polyacrylamide wells. The gel was stacked at 4%, separated at 12%, and run with a Bio-Rad Miniprotein 3 unit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 60 and 120 V for stacking and separating the gel, respectively. The gels were stained with 0.1% (w/v) Coomassie Brilliant Blue R250 staining solution for 2 h and de-stained with decolorization solution overnight. The protein standard with weight sizes ranging from 10 to 250 kDa was used as a marker. The gels were then scanned with Image Scanner III (GE Healthcare Biosciences, Uppsala, Sweden).

The filets were fixed in 2.5% glutaraldehyde overnight, sequentially dehydrated with a gradient of 30, 50, 70, and 90% ethanol; freeze-dried for 48 h, and photographed under a scanning electron microscope (Image Scanner III, General Electric Company, Schenectady, NY, USA) after gold spraying by ion sputtering (12 (link)).

The proteins were stained with CBB G-250 solution (Blue Silver) as previously described (
Candiano et al., 2004 (link)
). An ImageScanner III was used to digitize the gels, and the images were managed using LabScan 6.0 software (both from GE Healthcare). The images were analysed using ImageMaster 2D Platinum 6.0 software (GE Healthcare). The spots were analysed based on their area, volume and intensity, as well as distribution similarity among the triplicates. The student's t test was used, performed automatically by the software.

Tissue sample from human aortic valve was powdered under liquid nitrogen using a pestle and mortar and then lysed in sample lysis buffer (7 M urea, 2 M thiourea, 100 mM DTT, 4% CHAPS, 2% ASB-14, 1 mM Na3VO4, 1 mM NaF, 1% Protease Inhibitors). Samples were left for 15 min in ice, vortexing every 5 min, and centrifuged at 2500 ×g for 20 min. Total protein content of lysate was determined using a Bio-Rad protein assay. Twenty micrograms of sample were resolved by SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed with Anti-HGD or Anti-GAPDH antibodies, followed by Anti-Rabbit HRP-conjugated antibody. Detection was obtained by ImmunoStarHRP (Bio-Rad, Segrate, Milan, Italy); images were acquired using ImageScannerIII (GE Healthcare, Milan, Italy) and analyzed by ImageQuantTL (GE).

Transfected cells were flow sorted according to GFP status (Influx, BD Biosciences) then 400, 200 and 100 GFP positive or negative cells were seeded into 10 cm dishes and incubated for 2 weeks. Colonies were washed twice with PBS followed by fixation with neutral buffered formalin, 6% v/v for 30 minutes followed by overnight staining with 0.5% methylene blue, on a rocking platform. Plates were washed in cold tap water until the water ran clear, drained and allowed to dry. Plates were scanned (GE ImageScanner III) and colonies >50 cells were counted using Fiji (ImageJ2) software. Plating efficiency and surviving fractions were calculated as follows: Plating efficiency (PE) = number of colonies counted / number of colonies plated. Surviving fraction (SF) = PE sample / PE control.

Mustard seed extracts normalized to equal amounts of protein (10 µg) were subjected to SDS-PAGE under reducing and non-reducing conditions using pre-cast Any KD TGX gels (Bio-Rad Laboratories, Hercules, CA, USA) according to Laemmli [26 (link)]. The soluble extracts were mixed with an equal volume of Laemmli sample buffer with 5% (v/v) of β-mercaptoethanol (β-ME) and boiled for 5 min. Alternatively, electrophoresis was performed under non-reducing conditions by omitting the addition of β-ME. The gels were run at a constant voltage of 150 V for 90 min using TGS buffer (25 mM Tris, 192 mM glycine, and 0.1% SDS) in a Criterion cell (Bio-Rad). A molecular weight standard (Precision Plus Protein Standard) was included on each gel. After electrophoresis, gels were stained with Coomassie Brilliant Blue. Images were acquired by scanning stained gels using an Image Scanner III (GE Healthcare, Salt Lake City, UT, USA) operated by LabScan 6.0 software (GE Healtcare). For image and densitometry analysis, the Image Quant TL 7.0 Software (GE Healtcare) was used.