Emfc 6 ultramicrotome

Cryogenically fractured surfaces of tensile specimens were observed by scanning electron microscopy (SEM) after gold coating. A Hitachi S-2700 electron microscope (Fukuoka, Japan) was used at an accelerating voltage of 15 kV.
The nanostructure was analyzed by transmission electron microscopy (TEM). The samples were obtained from injection-molded specimens which were ultrathin sectioned at ~100 nm using a Leica EMFC 6 ultramicrotome equipped with a diamond knife. The micrographs were taken in a Tecnai G2 20 twin apparatus (FEI, Waltham, MA, USA), operating at an accelerating voltage of 200 kV.

The nanostructure was analyzed by transmission electron microscopy (TEM). The samples were obtained from injection-molded specimens and ultrathin-sectioned at ~100 nm using a Leica EMFC 6 ultramicrotome (Wetzlar, Germany) equipped with a diamond knife. The micrographs were obtained in a Tecnai G2 20 twin apparatus (FEI, Waltham, MA, USA), operating at an accelerating voltage of 200 kV.

To detect IgM binding to merozoites, parasites were tightly synchronized before late stage schizonts were purified using 70% Percoll, put back into culture containing RPMI + Albumax II with the addition of 20% human serum or 0.125 mg/ml purified human IgM (Sigma). IgM was detected using an Alexa Fluor 488 goat anti-human IgM μ chain antibody (Molecular Probes; preadsorbed against human IgG) at 1:1000. The slides were viewed on a Zeiss Axioplan 2 imaging system with Plan Apochromat 100×/1.4 oil immersion objective. Images were captured using Axiovision 4.6.3 software and edited using Adobe Photoshop.
For immunogold labeling, merozoites were fixed in 4% paraformaldehyde in 0.1 m phosphate buffer at pH 7.4 for 1 h at room temperature, rinsed three times in buffer, and infiltrated with 1% and then 10% gelatin before immersing in 2.3 m sucrose in phosphate buffer overnight at 4 °C for cryoprotection. Frozen samples were prepared by mounting onto aluminum pins and rapidly immersing in liquid nitrogen in preparation for ultrathin 80 nm sectioning on a Leica EM FC6 ultramicrotome. Ultra thin sections were labeled as per Tokuyasu (62 ), with a rabbit anti-human IgM antiserum (Abcam) diluted 1:25, and detected with 10-nm protein A gold. Imaging was performed on an FEI 120kV Spirit Biotwin with a Tietz F4.15 CCD camera.