Rat igg2a isotype control

To test the role of T-cells in the cross-protection induced by FluMist vaccine, mice were vaccinated with FluMist on days 0, 14, and 28. Mice were challenged with 100 × LD₅₀ PR8 influenza virus on day 42 post-vaccination. On days 1 and 3 before challenge, and day 1 after challenge, groups of mice were injected intraperitoneally with rat anti-mouse antibodies: 100 μg anti-CD4+ antibody, 100 μg anti-CD8a+, 100 μg CD4+ and 100 μg CD8+ antibodies, or 100 μg rat IgG2a isotype control (BD Pharmingen). After viral challenge, mice were monitored daily for clinical symptoms and survival.

Brains were removed from deeply anesthetized and exsanguinated mice, frozen in liquid nitrogen and sectioned in a cryostat. Parallel brain sections (20 μm thick) were fixed 45 min in 4% paraformaldehyde for CD4 immunostaining or 4 min in 4% phosphate buffered formaline, pH 7.4, followed by 2 min in 50%, 100%, and 50% acetone, respectively, and finally air dried for CD8a immunostaining. Sections were then rinsed in 0.5 % triton X-100 diluted in 0.05M trizma base buffered saline (TBS+T), pH 7.4, and incubated with 10% fetal calf serum (FBS) in TBS+T before incubation overnight at 4°C with purified rat-anti-mouse CD4 IgG2a (2.5 μg/mL) (553647, BD Pharmingen) or purified rat-anti-mouse CD8a IgGa (7 μg/mL) (MCA1108GT, AbDSerotec) diluted in 10% FCS in TBS+T. Parallel sections were incubated with rat IgG2a isotype control (2.5–7 μg/mL) (BD Pharmingen). After a rinse in TBS+T, endogenous peroxidase activity was blocked with 1.9% H₂O₂ in TBS, and sections were rinsed in TBS+T before and after incubation with biotinylated goat anti-rat IgG antibody (5 μg/mL) (BA9400, Vector Laboratories Inc) and HRP-conjugated streptavidin (1:300) (RPN1231V, GE Healthcare), respectively, diluted in 10% FCS in TBS+T. After a final rinse in TBS+T, the colour signal was developed in 0.05% 3.30-diaminobenzidine tetrahydrochloride and 0.003% H₂O₂ in TBS. Finally, sections were coverslipped with Depex.