The flies were immersed in fixing solution (3.38 ml of 37% formaldehyde (Merck), 0.5 ml of 1 M Na2PO4 (pH 6.8), 5 ml of octane, and 1.12 ml of water) for 20 min. Fixed heads were separated from the body in a phosphate buffer solution and put into a second fixing solution (0.43 ml of 37% formaldehyde (Merck), 0.4 ml of 1 M Na2PO4 (pH 6.8), and 3.17 ml of water) for 90 min at 4°C.
Embryos were washed in PBT and put into equilibration buffer (Roche) for 1 hour.
Approximately 25 mg of muscle and 500 µl of phosphate buffered saline was placed in a Medicon (BD Biosciences) and put into the Medimachine for 20 s.
Quadriceps femoris muscles of immunized mice were dissected, minced and put into 5 ml of digestion buffer before incubation at 37°C for 30 min.
Briefly, these sections were incubated with 1% H2O2 to inactivate endogenous peroxidase and put into 10 mM sodium citrate buffer solution to recover antigen.
Briefly, 100 mg of frozen tissue was thawed and put into 900 μl of dissociation buffer containing 25 mM HEPES (pH 7.2), 0.3 M sucrose and 53.6 μg Liberase Blendzyme 2 (Roche, Penzberg, Germany).
Following perfusion, a 2 cm spinal cord segment, with the injured site at the middle, was removed and put into 25% sucrose in phosphate buffer at 4°C until it sank.
Fifteen samples from each stage of tissue culture were randomly selected and put into 3% glutaraldehyde in 0.1 M cacodylate buffer for 90 min at room temperature.
The liver was weighted and put into a manual glass homogenizer with lysis buffer (20 uL/mg), which was heated and then centrifuged to obtain supernatant for future enzymatic assays, according to the manufacturer-supplied instructions.
These advances are critically reviewed and put into perspective.
