Anti rabbit igg fluorescein isothiocyanate fitc

AMSCs were grown in Dulbecco’s modified Eagle’s medium (L-DMEM), IMDM, DF12 (Life Technologies, San Diego, CA, USA), with platelet-derived growth factor BB (PDGF-BB, Sigma, St. Louis, MO, USA). The following antibodies were used: CD11a, CD29, CD31, CD34, CD44, CD45, CD73, CD105, CD106, CD166, CD184, HLA-ABC, HLA-DR (BD Biosciences, San Jose, CA, USA); anti-rabbit IgG- fluorescein isothiocyanate (FITC; Sigma), anti-mouse IgG-FITC (Sigma). Jagged-1 (Calbiochem, San Diego, CA); Jagged-2 (Chemicon, Temecula, CA); Delta-1(Santa Cruz Biotechnology, CA).

Nymphs of RCB were reared for 48 h on artificial diet supplemented with predetermined LC₅₀ of CEA. Midguts were dissected from the target insects and embedded in the water soluble embedding medium at −21 °C, and cross-sections of 15–20 µm were made using a Cryo microtome (Leica, Wetzlar, Germany). Fixation of midgut sections was performed using formaldehyde followed by extensive washing with 1× Phosphate buffer saline (PBS, pH 7.2). The sections were maintained in 2% buffered glycine followed by washing with 1× PBST (PBS with 0.2% tween20) at room temperature. Overnight blocking of sections was carried out in 2% buffered bovine serum albumin (BSA, Sigma-Aldrich) at 4 °C before washing in 1× PBS (pH 7.2). Midgut sections were then incubated with anti-CEA antibody (1:1000) for 2 h, followed by washing with 1× PBST and incubation with anti-rabbit IgG-Fluorescein isothiocyanate (FITC, Sigma-Aldrich) conjugate (1:2000) for 2 h at room temperature. The sections were then mounted in Keiser’s albumin and observed under a TCS SP5 confocal laser scanning microscope (Leica, Germany) using the excitation and emission ranges suited for FITC. Identical experimental setups were made for control insects reared on artificial diet without CEA.