Texas red conjugated goat anti mouse igg antibody

At different time points, consecutive cryosections of 16 μm were made of the frozen explants. The frozen sections were mounted on 3-aminopropyltriethoxysilane (Sigma-Aldrich, St. Louis, MO) coated slides. They were fixed in 100% methanol for 20 min at −20°C, and then washed with DPBS. Late viral proteins were stained with biotinylated equine polyclonal anti-EHV1 IgG antibody (1:20 in DPBS) (van der Meulen et al., 2003 ), followed by streptavidin FITC (1:200 in DPBS) (Molecular Probes, Eugene, OR). Subsequently, the BM of the explants was stained by incubation with a mouse monoclonal anti-collagen VII IgG1 antibody (clone LH7.2; 1:50 in DPBS) (Sigma-Aldrich, St. Louis, MO), followed by a Texas Red®-conjugated goat anti-mouse IgG antibody (1:200 in DPBS) (Molecular Probes, Eugene,OR). Antibodies were incubated for 1 h at 37°C. Determinations of viral plaque numbers and plaque latitude were based data obtained from 50 consecutive cryosections.

At 24 hpi, fifty consecutive cryosections of 16 μm were made of the frozen explants derived from three horses. The frozen sections were mounted on 3-aminopropyltriethoxysilane coated slides. They were fixed in 100% methanol for 20 min at -20°C, and then washed with DPBS. Late viral proteins were stained with biotinylated equine polyclonal anti-EHV1 IgG antibody (1:20 in DPBS) (van der Meulen et al., 2003 ), followed by streptavidin FITC (1:200 in DPBS) (Molecular Probes). Subsequently, the basement membrane of the explants was stained by incubation with a mouse monoclonal anti-collagen VII IgG1 antibody (clone LH7.2; 1:50 in DPBS) (Sigma-Aldrich), followed by a Texas Red^®-conjugated goat anti-mouse IgG antibody (1:200 in DPBS) (Molecular Probes). Antibodies were incubated for 1 h at 37°C. Cell nuclei were counterstained with Hoechst 33342 (10 μg ml^-1). Determinations of viral plaque numbers and plaque latitude were data obtained from 50 consecutive cryosections.