Bound antibodies were detected with polyvalent, peroxidase-conjugated goat anti-human Ig and visualized by incubating the nitrocellulose strips in chemiluminescent reagents (NEN Life Science Products Inc., Boston, MA, USA) and exposing to Kodak XAR-5 films.
For CK7 and caspase 3, signal was developed by the polyvalent LSAB-peroxidase Dako Kit (Dako).
Secondary antibodies were polyvalent horse radish peroxidase-conjugated goat anti-human IgG, anti-mouse (IgG) or anti-rabbit IgG, IgA, and IgM (Zymed, San Francisco, CA).
Homemade indirect enzyme-linked immunosorbent assay (ELISA) was used for detection of serum Abs employing goat anti-human polyvalent (anti-IgA/IgM/IgG) horseradish peroxidase- (HRP-) conjugated secondary Ab as detection reagent (Sigma-Aldrich Co. St . Louis MO, USA).
Serum was then added to the wells, followed by hyperimmune rabbit Ebola polyvalent antiserum and then peroxidase-conjugated goat antibodies against rabbit IgG.
Sample extracts (see above) were then added to the wells, followed by hyperimmune rabbit Ebola polyvalent antiserum and then peroxidase-conjugated goat antibodies against rabbit immunoglobulin G (IgG).
The plate was washed and peroxidase-conjugated goat anti-human polyvalent immunoglobulins (IgG, IgA, and IgM) (1∶10000) or peroxidase-conjugated goat anti-human IgG (1∶12000) antibody (Sigma-Aldrich, UK) in PBS was added to each well for 1 h.
The filters then were incubated overnight with E. coli lysate-depleted sera of investigated subjects (1∶500 dilution in PBST), washed three times with PBST, and incubated with a anti-human polyvalent immunoglobulins (IgG, IgA, and IgM) peroxidase-conjugated secondary antibody (Sigma-Aldrich, UK) at a dilution of 1∶10000 with PBST for 1 h.
Detection was achieved via a biotin-conjugated polyvalent antibody with an avidin-biotin-peroxidase reagent (Scy Tek, Logan, USA) and diamino-benzidine staining.
Sections were incubated with mouse monoclonal antibodies against FVIII (Dako) or αSMA (Abcam, Cambridge, UK) at room temperature for 1 h, rinsed in TBS Tween, and incubated with secondary antibodies (UltraSens Biotinylated Goat Anti-polyvalent RTU; ImmunoLogic) followed by streptavidin peroxidase (UltraSens Streptavidin Peroxidase RTU; ImmunoLogic) for 10 min each.
