The sections were blocked with normal rabbit serum (in case of cetuximab) or with normal swine serum (in case of anti-P-gp) and subsequently stained with cetuximab 10 μg/mL (EGFR) or anti-P-gp 5 μg/mL.
Tissue sections were pre-incubated for 10 minutes with normal swine serum, followed by incubation for 1 hour with C4BP antibodies.
Following fixing, cells were washed in TBS (50 mM Tris-HCl, 150 mM NaCl pH 7.4) and blocked using 5% normal swine serum diluted in TBS.
Cell cultures or myofibres were fixed in 4% paraformaldehyde/PBS for 10 minutes, permeabilised with 0.5% (v/v) Triton X-100 in PBS and then blocked using 10% (v/v) goat serum and 10% (v/v) swine serum in PBS.
Sections were washed (6×10 min) in Tris-SMB buffer with 0.5% of Triton X100 (Tris-SMB-TX), then preincubated with 5% Normal Swine Serum (Dakopatts a/s, Glostrup, Denmark) for one hour.
For control of double immunostaining (Figure S1A) we used Tris-HCl buffer with 5% swine serum instead of GABA antiserum (Fig. S1B) or AmOA1 antiserum (Fig. S1C) on two consecutive sections.
Slides were then incubated for 15 18 h at 4°C with the primary antibodies (rabbit anti-SP; BP823, DPC Biermann GmbH, Hiddenhausen, Germany; diluted 1∶100; rabbit anti-MHV-68, diluted 1∶2000) in TBS with 20% swine serum.
Sections were retreated with citrate as above, blocked 5% normal swine serum (Jackson) and labeled for 3 hours with c-KIT specific antibody followed by labeling with a AP-conjugated secondary antibody.
Briefly, the paraformaldehyde-fixed myofibres were permeabilised with 0.5% (v/v) Triton X-100 in PBS for 6 mins and then blocked using 10% (v/v) goat serum (DakoCytomation) and 10% (v/v) swine serum (DakoCytomation).
Myofibres were fixed in 4% paraformaldehyde/PBS for 10 minutes, permeabilised with 0.5% (v/v) Triton X-100 in PBS and then blocked using 10% (v/v) goat serum and 10% (v/v) swine serum (DakoCytomation, Ely, UK) in PBS.
JEV-positive swine serum was used as a positive control.
