After washing three times in PBS for 30 min, the materials were postfixed for 2 h in 1% osmium tetroxide in 0.1 M PBS (pH 7.4) at 4°C, and rinsed four times with distilled water (pH 7.0).
The materials were macerated in 1 mol/L hydrochloric acid at 60°C for 10 min. After washing three-five times to eliminate residual hydrochloric acid and staining with carbol fuschin for 1 min, the materials were squashed for observation in 45% acetic acid.
Briefly, brain tissue was incubated in 1% sodium citrate for 10 min at room temperature and transferred to methanol-acetic acid 3∶1 solution for 4 min. The material was disrupted in 100 µl 60% acetic acid and dropped onto clean slides and dried.
The synthesis can be done in pure water (containing catalytic amounts of NH4F) at room temperature within a reaction time of 2 min. The material structures derive from lamellar structures.
The fraction containing the internalized receptor, referred to as the endosome fraction, was obtained by sucrose density gradient centrifugation of the 16,000×g supernatant on a 0.67 M and 1.05 M discontinuous sucrose gradient at 100,000×g for 75 min. The material collected from the interphase between the 0.67 and 1.05 M sucrose is referred to as the endosome fraction [12].
Then, the excess of acetic acid was removed, and a drop of acetic acid:water:lactic acid mixture in 3 2 1 (v/v) ratio was added and rested for 4 min. The material was then placed on a slide, spread, and squashed.
In contrast, the extreme alkaline pretreatment (that is, pH 13, 140°C, 10 min) prepared the material well for enzymatic liberation of glucose and xylose as the enzyme catalyzed monosaccharide release increased significantly with increased enzyme dosage.
In under 20 min, the hybrid material could be obtained in a one-pot reaction.
After ultrasonication for 20 min, the insoluble material was separated by centrifugation for 15 min at 16,000 g.
The fermented broths were centrifuged at 12,587 × g at 4°C for 20 min, the precipitated material was discarded, and the supernatant was filtered through a 0.22-μm membrane and further concentrated by freeze-drying.
