Retsch tissue lyser mm300

Organs (brain, spinal cord, ovaries, uterus, placenta, embryonic head) were removed under aseptic conditions from sacrificed mice (n = 8–10). The organs were immediately frozen and subsequently thawed, weighed, and homogenized using 0,1 g of organ material with 1 ml PBS in a microtube (Retsch Tissue Lyser MM300, Qiagen GmbH, Hilden, Germany). Tubes were centrifuged for 1 min at 1.500 rpm and 4 °C. Supernatants were taken and stored at −80 °C. Viral titers in organ supernatants were determined by plaque assay as described before [39 ] and indicated in PFU per 1 g organ material.

Organs of chicken embryos were weighed, freeze-thawed three times and homogenized with PBS in a microtube for 45 s at lowest level (Retsch TissueLyser MM 300; Qiagen GmbH, Hilden, Germany). After centrifugation (1 min; 1500 rpm; 4 °C), supernatants were stored at −80 °C.
Virus infectivities were determined via routine plaque assays performed in duplicate [18 (link)]. In 6-well-plates, confluent CEF monolayers were infected with serial 10-fold dilutions of the organ supernatants. After two hours at 37 °C, the cells were washed with PBS. After washing, cells were incubated at 37 °C for two days with virus growth medium [18 (link)]. The infected cells were fixed with acetone-methanol and incubated with polyclonal rabbit anti-vaccinia antiserum (1:1000, Acris, BP1076), followed by peroxidase-conjugated goat anti-rabbit antibody (1:5000, Jackson ImmunoResearch, 111-035-035). Infectious foci were visualized with TrueBlue, counted, calculated, log10-transformed, and indicated in IU/g organ.