Image pro premier 9

Agar (0.35%, 1 mL) in culture media was added to the bottom layer of each well in a 12‐well plate. For the top layer in each well, 1 × 10³ cells were resuspended in 1 mL of a mixture comprising 0.2% agar and RRMI 1640 media. The cells were incubated at 37°C with 5% CO₂ for 2 weeks, after which the colonies were stained with crystal violet, photographed by inverted phase‐contrast microscopy (Leica, Wetzlar, Germany) and counted using ImagePro premier 9 (Image‐Pro Premier 9.0, Media Cybernetics, MD, USA).

Liver paraffin-embedded sections were deparaffinized, rehydrated, and then stained with haematoxylin-eosin, following standard protocols. In detail, haematoxylin-eosin staining was used to evaluate liver morphology and also to measure the hepatocyte nuclear area [13 (link), 40 (link)]. The hepatocyte nuclear area was calculated using a computerized image analyzer (Image Pro Premier 9.1, Media Cybernetics Inc., Rockville, USA) evaluating 20 randomly chosen liver fields per experimental animal. Two blinded investigators performed the morphometrical analysis and their evaluation was assumed to be correct if the values were not significantly different. If there was disagreement concerning the interpretation, the case was reconsidered to reach a unanimous agreement.

Serial paraffin gastrocnemius sections were stained with hematoxylin-eosin and the sections were then observed with a light microscopy at final magnification of 400× (Olympus, Hamburg, Germany). Feret’s diameter of 50 myotubes for each animal gastrocnemius was determined using an image analyzer (Image Pro Premier 9.1, Media Cybernetics, Rockville, MD, USA). Two blinded investigators performed the morphometrical analysis and their evaluation was assumed to be correct if the values were not significantly different. If there was disagreement concerning the interpretation, the case was reconsidered to reach a unanimous agreement.

Reactive oxygen species were analyzed using fluorescent probe dihydrorhodamine 123 (Invitrogen; Thermo Fisher Scientific, IL, United States) applied to frozen heart sections. The sections were mounted and observed with fluorescent microscopy (i50 Eclipse, Nikon, Hamburg, Germany) at a final magnification of 400×. Twenty random fields from a total of five non-consecutive sections per animal were analyzed, and the fluorescence staining was calculated using an image analyzer (Image Pro Premier 9.1, Media Cybernetics, Rockville, MD, United States) and expressed as arbitrary units (AU). Two blinded investigators performed the analysis, and their evaluations were assumed correct if the values were not significantly different. If there was disagreement concerning the interpretation, the case was reconsidered in order to reach a unanimous agreement.

Gastrocnemius samples were fixed in 4% buffered paraformaldehyde for 24 h, dehydrated in progressive ethanol solutions, xylene and embedded in paraffin wax, following the standard procedures. Subsequently, 7 mm-thick paraffin sections were cut with a microtome and so the serial paraffin sections were dewaxed in xylene, rehydrated through decreasing scale of ethanol and stained with haematoxylin-eosin, following standard protocol. Then the sections were observed with an optical light microscope (Olympus, Deutschland, Germany) at the final magnification of 200×. Digital images of gastrocnemius were captured and the Feret’s diameter (expressed in µm) of 50 myotubes for each animal muscle was determined using an image analyser (Image Pro Premier 9.1, Media Cybernetics, Rockville, MD, USA) by two observers blinded to the treatment, whose evaluation was assumed to be correct if the values were not significantly different. In case of dispute concerning interpretation, the case was reconsidered to reach an agreement [73 (link),74 (link)].

Coronary artery segments of 1 to 2 mm from domestic or Ossabaw pigs were formalin-fixed, paraffin embedded, and cross-sectioned. The sections were deparaffinized with xylene, hydrated, and subjected to the standard acidic antigen retrieval procedure. The primary goat K_v7.4 antibody (dilution factor: 1:100, Santa Cruz Biotechnology, Dallas, TX) or rabbit K_v7.5 antibody (dilution factor: 1:50, Alomone Labs, Jerusalem, Israel) were added to the prepared sections. After overnight incubation at 4°C with the corresponding primary antibody, the sections were then incubated with the secondary biotin-conjugated antibody (dilution factor 1:1,000, The Jackson Laboratory, Sacramento, CA). After the secondary antibody was removed by multiple washes, the sections were treated with horseradish peroxidase-conjugated to streptavidin. The color was developed using diaminobenzidine and appeared brown. The color density of staining was analyzed using the DAB analysis module of Image-Pro Premier 9.1 (Media Cybernetics Inc., Warrendale, PA). The ratio of the background-corrected intensities of media/intimal layer and the adventitia layer staining was calculated to quantify the relative expression levels of K_v7 proteins. No staining was observed in “no primary antibody” controls (S1 Fig).