Alexa fluor 488 protein a conjugate

OURT and GRG isolates have been described previously^{10 (link), 12 (link)}. The Vero cell-adapted strain, BA71V^{40 (link)}, was used as antigen for the IFA assay. All viruses were generously provided by Dr. Linda Dixon (Pirbright Institute, United Kingdom). All in vitro and in vivo work involving live ASFV was conducted within biosafety level 3 facilities at the AAHL. GRG and OURT were cultured in primary porcine bone marrow (PBM) cells using RPMI media supplemented with glutamine, HEPES, penicillin and streptomycin, Fungizone and 12.5% fetal calf serum (FCS, Serana)^{41 (link), 42 (link)}. After 4 days incubation, inoculum was removed, cells were washed, then fixed with cold acetone/methanol for 30 min at room temperature. Cells were washed three times with PBS and incubated for 1 hr at 37 °C with an in-house rabbit anti-ASFV p32 polyclonal antibody diluted in PBS with 1% BSA and 0.005% Evans Blue. Following three washes, Alexa Fluor 488 protein A conjugate (ThermoFisher) was added to each well and incubated for 30 min at 37 °C. Following a final wash step, fluorescence was visualized with an IX71 Olympus inverted fluorescence microscope. TCID₅₀ was calculated according to the method of Reed and Muench^{43 (link)}.

Cryo-sections of 10 μm from non-treated mice bearing WEHI-164 tumor were fixed in ice-cold acetone for 10 min and blocked for 1 h in FBS/BSA (20%/2% solution in PBS). EDB expression was detected by using L19-TNF^WT or L19-TNF^I97A respectively (10 μg/mL in PBS/2% BSA). Alexa Fluor 488 protein-A conjugate (Thermo Fisher; 1:200) was used as a secondary detection antibody. Vessels were stained with a rat anti-mouse CD31 (BD Pharmigen; 1:200) and detected with a donkey anti-rat Alexa Fluor (Invitrogen; 1:500). Slides were mounted with Dako fluorescent mounting medium (Agilent) and sample representative pictures were taken with a 10x objective lens using an Axioskop2 mot plus microscope (Zeiss; ETH, Switzerland).