Goat anti rabbit hrp conjugate

Rabbit polyclonal antibodies against EBOV NP, VP35, and VP30 proteins have been described previously (16 (link)). Mouse polyclonal antibody against EBOV VP40 protein was a gift from Zhong Huang (Institut Pasteur of Shanghai). Mouse monoclonal antibody against β-actin (catalog no. AM1021B) was bought from Abgent. The following secondary antibodies were used: donkey anti-mouse Alexa Fluor 555 conjugate (catalog no. A31570; Thermo Fisher Scientific), goat anti-mouse Alexa Fluor 555 conjugate (catalog no. A21422; Thermo Fisher Scientific), goat anti-mouse horseradish peroxidase (HRP) conjugate (catalog no. 115-035-003; Jackson ImmunoResearch), and goat anti-rabbit HRP conjugate (catalog no. 31460; Thermo Fisher Scientific).

BALB/c mice (6-day old, n = 3) were pretreated with sterile PBS, CPMO1v or sCPMO1v and subjected to CHIKV infection as described above. At day 7 p.i., mice were sacrificed and their limbs and liver were harvested and formalin fixed, dehydrated and paraffin embedded. Sectioning of tissue samples was done at 4 μm and samples were routinely stained with haematoxylin and eosin (H&E). For IHC, tissue samples were labeled with primary rabbit E2 antibody diluted to 1:100, followed by a secondary goat anti-rabbit HRP conjugate (Thermoscientific). The assay was subsequently performed using automated Bond-Max System (Leica Biosystems, Germany) based on a customized protocol with several steps of dehydration and differentiation. Final slides were then mounted with PermountTM mounting medium (Fisher Chemical, UK) and viewed under Olympus microscope. Images were captured at 400× magnification to visualize CHIKV antigen.