Tp1020

After mice were sacrificed by cervical dislocation, the organs were weighed and rinsed with physiological saline. A portion of the epididymal adipose tissue was fixed in 4% paraformaldehyde for 48 h at 4 °C. The remaining organs were stored at −70 °C. Fixed epididymal adipose tissue was dehydrated by an automated tissue processor (TP1020; Leica Biosystems, Nussloch, Germany) using a series of graded ethanol solutions, embedded in paraffin and cut into 8-μm tissue sections. Sections were stained with hematoxylin and eosin (H&E), and adipose tissue cell morphology was recorded by optical microscopy. The size of adipose tissue cells was estimated by Adiposoft software (National Institutes of Health, Bethesda, MD, USA).

Once the muscle tissues were obtained, they were processed by a pathologist using a validated protocol for hematoxylin–eosin staining. The muscle sample was fixed from 24 to 48 h in 10% neutral buffered formalin. The samples were dehydrated in increasing ethanol solutions, cleared in xylol, and embedded in paraffin on an automatic tissue processor (Leica Biosystems, TP1020). Subsequently, 3 μm sections were obtained and stained with the conventional hematoxylin and eosin technique, and then evaluated by light microscopy (Microscope Carl Zeiss Axio Lab.A1).
Patients were grouped according to histological features in connective tissue increased, diffuse and multifocal atrophy, perifascicular atrophy, perivascular inflammatory infiltrate, endomysial inflammatory infiltrate, degenerated fibers, necrotic fibers, basophilic fibers, edema and bleeding, muscle fiber size variation, endothelial hyperplasia, and tumefaction.

At the end-point of each experiment animals, 200 μl of retro-orbital blood was collected from anesthetized animals and afterwards euthanasia was performed by cervical dislocation. For tissue array, necropsy of animals was performed according to the methods described by the Jackson laboratory. The order of extraction was the following according to the order of tissue autolysis: liver, pancreas, kidney, brain and muscle. Samples extracted were collected in Optimal Temperature Compound (O.C.T) and flash frozen fresh until further use. Before staining, O.C.T. included samples were cut using cryostat (Leica Biosystems CM1520). Alternatively, biological tissues were fixed in 10% neutral buffered formalin (NBF) (20:1 v/v). The 10% NBF was replaced by fresh fixative to eliminate blood and feces from the fixative. After 24h of fixation, tissues were placed in 50% alcohol and processed and embedded in paraffin with an automated tissue processor (Leica Biosystems TP1020, Barcelona, Spain) and an embedding center (Leica Biosystems EG1150H, Barcelona, Spain). Tissues were oriented properly previous to the final embedding in paraffin.