Vectastain universal abc ap kit

The monoclonal antibody (mAb) G3 (IgG1) against tryptase was purchased from Chemicon (Temecula, CA), a rabbit polyclonal antibody against KIT (CD117) from Dako (Glostrup, Denmark), biotinylated anti-rabbit IgG, anti-mouse IgG and Vectastain Universal ABC-AP Kit from Vector Laboratories (Burlingham, CA), and 3-amino-9-ethylcarbazole (AEC) from Sigma (St.Louis, MO, USA). Imatinib was kindly provided by Dr.E.Buchdunger and Dr.P.Manley (Novartis Pharma AG, Basel, Switzerland). Recombinant human (rh) stem cell factor (SCF) was from Strathmann Biotech (Hannover, Germany) and rh interleukin-6 (IL-6) from Novartis Pharma AG. RPMI 1640 medium and fetal calf serum (FCS) were purchased from PAA laboratories (Pasching, Austria) and a histamine radioimmuno-assay (RIA) from Immunotech (Marseilles, France). Serum and cellular tryptase levels were measured by fluoroenzyme-immunoassay (FEIA, Thermo Fisher Scientific, Uppsala, Sweden). The detection limit for total (alpha+beta-type) tryptase in this assay was 1 ng/mL. The median serum tryptase level in healthy controls amounts to 5.6±2.8 ng/mL (range: 0-15 ng/mL) [48 (link)].

BHK-21 cells were infected with MVA 1974 at a multiplicity of infection of 0.05. Infected cells were transfected with pTP-GP using Lipofectamine (Life Technologies) as directed by the manufacturer. The resulting recombinant MVA-GP was serially plaque-purified 4 times in BHK-21 cells, based on green fluorescent protein (GFP) expression. MVA-GP was amplified on BHK-21 and CEF cells, purified by sucrose cushion centrifugation [30] and titrated by plaque assay [26] on BHK-21 cells, prior to use in in vivo studies. Plaques were visualised using GFP fluorescence or by immunostaining [26] with rabbit anti-Vaccinia antibody (AbD Serotec, UK) and Vectastain Universal ABC-AP kit (Vector Laboratories, USA). Genomic DNA from infected cells was extracted using the Wizard SV genomic DNA purification system (Promega, USA) and used as a template in PCR with AccuPrime Taq DNA polymerase High Fidelity (Life Technologies) for genotype analysis.