A chromogenic method using 3,3′-diaminobenzidine tetrahydrochloride (Sigma–Aldrich, St. Louis, MO, USA) as a substrate was also employed.

The intensities of the protein bands were analyzed and compared using the Scion Image software, version Alpha 4.03.2 (Scion Corporation, Selleck 3-Methyladenine Frederick, MD, USA), and the results were expressed as a percentage of the total content. In this assay, 40 brains were utilized to obtain the protein fraction that was separated using a Sephacryl S-400 gel filtration column (15 mL volume; Amersham Pharmacia Biotech, Uppsala, Sweden). The column was equilibrated with homogenization buffer and loaded with 3 mg of total protein in a volume of 400 μL. Elution fractions (150 μL) were analyzed by SDS–PAGE and Western blot. Myosin-Va solubility was assessed from protein extracts (Section 2.4), obtained homogenizing honey bee brains with or without 5 mM ATP, and centrifuging homogenates at 40,000g for 40 min at 4 °C. The supernatant fractions were analyzed by protein quantification,

SDS–PAGE and Western blot. The myosin-Va-enriched fraction was prepared using the initial fractionation steps of an established protocol for myosin-Va purification (Nascimento et al., 1996). Honey bee brains were homogenized in homogenization buffer at 4 °C, and centrifuged at 40,000g at 4 °C for 40 min. The salt concentration of this supernatant (S1) was increased to 0.6 M NaCl, and the solution was then incubated on ice for 1 h. The pellet (P2) and supernatant (S2) were separated by centrifugation of the salt-treated

Crizotinib cost S1 at 40,000g at 4 °C for 40 min. The fractions obtained were analyzed by total protein content, SDS–PAGE and Western blot. Brains were fixed in Carnoy solution (ethyl alcohol:chloroform:glacial acetic find more acid, 60:30:10 by volume) with 1.2% (w/vol) sodium sulfate for 90 min, dehydrated and paraffin-embedded. Eight-micrometer sections were incubated in cresyl violet solution (0.5% (w/vol) cresyl violet, 1 M sodium acetate, and 1 M acetic acid, pH 3.9) for 30 min or incubated in a solution containing 120 mM citrate buffer, 36% (w/vol) arabic gum, 100 mM hydroquinone and 0.08% (w/vol) silver nitrate for 30 min at 35 °C (Babb et al., 1991). The sections were then dehydrated and mounted with Permount (Fisher Scientific, Fair Lawn, NJ, USA). Brains were dissected, fixed in 4% paraformaldehyde, and paraffin-embedded (McLean and Nakane, 1974). Five-micrometer sections were cut and mounted on gelatin-chromium potassium sulfate (chromealum)-coated microscope slides. After antigen retrieval using 10 mM citrate buffer (pH 6.0), antibody detection in the tissue sections was performed according to Calabria et al. (2010) and Martins et al. (1999). Then, the sections were incubated with H2O2 in phosphate-buffered saline (PBS), pH 7.4, for 15 min, followed by a 4 h incubation in 0.02 M sodium phosphate buffer, pH 7.4, containing 450 mM NaCl, 0.