Visualizar Tese - Instituto de Biociências - Unesp

Andreo Fernando Aguiar(mATPase) histochemistry was performed using preincubation at pH 4.2, 4.5 and 10.6.Analyses revealed pure (Type I, IIA, IID and IIB) and hybrid muscle fibers (Type ICand Type IIC) based on their staining intensities (56) (Fig. 2). Muscle fiber–typepercentages were determined using Image Analysis System Software (LeicaQWin Plus,Germany).Fig. 2. Serial cross sections of plantaris muscle samples taken from a representativecontrol animal showing fiber-type distribution using myofibrillar adenosinetriphosphatase histochemistry after preincubation at pH 4.2 (A), 4.5 (B), and 10.6 (C). I,type I fiber; IIC, type IIC fiber; IIA, type IIA fiber; IID, type IID fiber; and IIB, type IIBfiber.Biochemical analyses. Myosin heavy chain (MHC) isoforms analysis wasperformed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in duplicate (maximum 5% of variation). Eight histological sections (12-mmthick) were collected from each sample and placed in a solution (0.5 mL) containingglycerol 10% (w/vol), 2- mercaptoethanol 5% (vol/vol), and sodium dodecylsulfate(SDS) 2.3% (w/vol) in a Tris/HCl buffer 0.9% (pH 6.8) (w/vol). The final solution wasshaken for 1 minute and heated for 10 minutes at 60ºC. Small amounts (8 µL) of theextracts were submitted to electrophoresis reaction (SDSPAGE 7–10%), using a 4%stacking gel, for 19 to 21 hours at 120V. The gels were stained with Coomassie Blue (6)and used to identify the MHC isoforms according to their molecular weight showingbands at the MHCI, MHCIIa, MHCIIx/d and MHCIIb levels (Figure 7A). The gels werephotographed and images were captured by VDS Software (Pharmacia Biotech).Finally, densitometry was performed using Image Master VDS Software (version 3.0),which determined the relative MHC isoforms content.49