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.