Similar protocols

Protocol publication

[…] Tissues were fixed in 4% paraformaldehyde overnight, followed by sinking in sequentially greater sucrose solutions (10%, 20%, and 30%). Fixed tissues were embedded in OCT compound (Sakura), and tissue blocks were frozen in 2-methylbutane (Sigma–Aldrich) chilled by dry ice. Eight micrometer sections were cut with a cryostat, air-dried, and fixed with cold acetone. Sections were stained with 1–5 μg/ml antibodies against Ly5.1, CD4, PNA, IgD, and ProLong Gold antifade reagents (Invitrogen) was added after washing. Images were captured on a Zeiss LSM 510 Meta confocal microscope, mounted on an Axiovert 100 M with automated XYZ control equipped with an argon laser with emissions at 458, 488, and 514 nm and two HeNe lasers with emission wavelengths at 543 and 633 nm. Image analysis was performed using Imaris suite (Bitplane, South Windsor, CT). [...] 96-well Polysorp microtiter plates (Nunc) were coated overnight with UV-inactivated WSN-GP33/66 in carbonate buffer. AP-conjugated goat anti-mouse IgG and IgA secondary antibodies were used for detection (Southern Biotech). ODs were converted to units based on standard curves with sera from C57BL/6 mice infected with influenza (Softmax Pro 3.1 software; Molecular Devices). […]

Pipeline specifications

Software tools Imaris, SoftMax Pro
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Virus Diseases
Chemicals Sirolimus