Application suite software version 4

Mature neurons were identified with Anti-NeuN antibody in control and AβO microinjected rats sacrificed on days 15 and 30 after stereotaxic surgery (n = 4, per group and time). For degenerated neurons, the FJB staining was evaluated in control and AβO groups at 18 h, 72 h, and 7 days post-surgery (n = 4, per group and time).
Quantification of NeuN and FJB positive cells was realized with 8 µm-thick sagittal sections obtained from injury (2.5–2.7 mm lateral from bregma), one out every 10 sections was evaluated. Cell count was performed on the Leica Application Suite software version 4.0 (Leica Microsystems, Wetzlar, Germany) on a 265 × 365 µm frame at 40× magnification, analyzing 3 adjacent fields on CA1 region and the same for dentate gyrus. The experimental groups were blinded in during the count and we considered positive cells with whole cell bodies within the counting frame. Average of positive cells per mm² were reported [80 (link),81 (link)].

Samples of the liver, pancreas, and epididymal adipose tissue were fixed with 10% formalin solution. After fixation, the specimens were dehydrated, embedded in paraffin, cut in a microtome to a thickness of 5 mm each, and stained with hematoxylin-eosin. An expert pathologist performed the histological analysis of the liver and pancreas. For the analysis of treatment effects on the hepatocytes, a scoring system was used [32 (link)]. In the evaluation of the architecture of the pancreas, there were changes in the Islets of Langerhans and pancreatic acini, and inflammation was observed [33 (link),34 (link)]. For the analysis of the adipocyte area of the epididymal adipose tissue, the images were initially taken using a LEICA DFC 495 digital camera system (Leica Microsystems, Wetzlar, Germany) integrated into a LEICA DM 5500B microscope (Leica Microsystems, Wetzlar, Germany), with a magnification of 20X. The images were analyzed using the LEICA Application Suite software, version 4.0 (Leica Microsystems, Wetzlar, Germany), and the mean area of 100 adipocytes per sample was determined [35 (link)].

Samples of the liver, pancreas, and epididymal adipose tissue were fixed with 10% formalin solution. After fixation, the specimens were dehydrated, embedded in paraffin, cut in a microtome to a thickness of 5 mm each, and stained with hematoxylin and eosin [41 (link),43 (link)]. An expert pathologist performed a blind histological analysis of the liver and pancreas and classified the samples according to a score system [45 (link),46 (link)]. Pancreas analysis followed the architecture of pancreas evaluation, according to changes in the islets of Langerhans [41 (link),43 (link),47 (link),48 (link)].
The adipocyte area of the epididymal adipose tissue was photographed by using a Leica DFC 495 digital camera system (Leica Microsystems, Wetzlar, Germany) integrated into a Leica DM 5500B microscope (Leica Microsystems, Wetzlar, Germany), with a magnification of 200x. The images were analyzed by using the Leica Application Suite software, version 4.0 (Leica Microsystems, Wetzlar, Germany), and a mean area of 100 adipocytes per sample was determined [49 (link)].