Rabbit anti np antibody

For immunostaining, infected Calu-3 cells were fixed with 4% PFA for 15 min. Following a washing step with PBS, cells were blocked and permeabilized overnight with 1% BSA in PBS (+0.2% Triton) and afterwards stained with a polyclonal rabbit anti-NP antibody (GeneTex, Cat. No. GTX135357, Irvine, CA, USA) for 24 h. Subsequently, cells were incubated for 1 h with a goat anti-rabbit-AlexaFlour488 (Invitrogen, Cat. No. A11008, Waltham, MA, USA) and finally stained with 4′,6-Diamidino-2-phenyl-indol –dihydrochlorid (DAPI) (Sigma Aldrich, D9542, St. Louis, MO, USA) for 10 min. For analysis, immunostaining was quantitative analyzed with a PerkinElmer VictorX4 reader (488 nm) (PerkinElmer, Waltham, MA, USA) and pictures were taken using a CTL-ELISPOT reader (Cellular Technology Ltd., Shaker Heights, OH, USA).

Lungs were collected at 5 dpi and they were fixed in 10% neutral buffered formalin. Sections (4 mm thick) were stained with hematoxylin and eosin (H&E) and examined through light microscopy. For immunohistochemistry, Deparaffinized, rehydrated tissue sections were incubated in antigen retrieval buffer for 15 min at 97°C and endogenous peroxidase was quenched using 3% H₂O₂ in methanol for 10 min (18 (link)). Then, tissue sections were stained with rabbit-anti-NP antibody (GeneTex, USA, GTX125989, 1:200). Goat antirabbit immunoglobulin conjugated to peroxidase (Maxim Bio, Fujian, China) was used as secondary antibody. Screening of sections was performed with an Olympus BX51 microscope coupled to a camera.