2.6 Histological analysis
The liver of the offspring (males and females) were sectioned, immediately immersed in 4% paraformaldehyde (PFA) fixative solution and buffered for 48 hours for subsequent histological processing. The material was embedded in paraffin and cut with a microtome, obtaining 4 µm thick sections. Slides were stained with hematoxylin & eosin (H&E) staining for image acquisition and histopathological analysis of liver histoarchitecture.
To analyze protein expression in an automated way, the technique of color morphometry was used, which was adapted to obtain the intensity and destruction of nuclear expression of the protein. Therefore, the H&E stained slides were submitted to the Axio Scan.Z1®(ZEISS, Jena, Germany) to capture images of all sample areas. After pre-processing these images with Adobe Photoshop CS6 v 13.0 ® (Adobe, San Jose, CA), in order to improve quality and separate images, they were analyzed with the Image Pro Plus v.4.5.0.29 (Media Cybernetics, Rockville, MD) software using the color morphometry tool. Initially, the software analyzed the total distribution of the white steatosis area in the tissue. With the help of the software, the steatosis area was marked in red. This method of marking a particular structure based on its color was called “mask”.
The software automatically provided the marked areas, quantified in square micrometers, through the following formula: (value of the white steatosis area/value of the image field area). Subsequently, the results were transported to an excel spreadsheet and quantified.