Tritc conjugated anti mouse igg

The cells were fixed with 4% paraformaldehyde in PBS buffer. After blocking with 20% AquaBlock (EastCoast Bio, North Berwick, ME, USA) for 30 min at room temperature, the cells were incubated overnight at 4 °C with a guinea pig anti-insulin antibody (1:100; Abcam, Tokyo, Japan), rabbit anti-C-peptide antibody (1:200; Cell Signaling Technology, Danvers, MA, USA), goat anti-Pdx1 antiserum (1:100; R&D system, Minneapolis, MN, USA), or mouse anti-Nkx6.1 antiserum (1:100; Developmental Studies Hybridoma Bank, Iowa city, Iowa, USA) and then for 1 h at room temperature with FITC-conjugated anti-guinea pig IgG (1:250; Abcam), Alexa Fluor 647-conjugated anti-rabbit IgG (1:250; Cell Signaling Technology), NorthernLights^TM NL493-conjugated anti-goat IgG (1:200; R&D system, Minneapolis, MN, USA) or TRITC-conjugated anti-mouse IgG (1:200; Sigma-Aldrich). Mounting medium for fluorescence with DAPI (Vector Laboratories, Peterborough, UK) was used for mounting. The percentage of insulin/C-peptide-positive cells was calculated based on the ratio of immunostaining-positive cells/DAPI-positive cells in 12 visual fields.

Before ending the experiment (30 min prior to sacrificing the mice), the tumor bearing animals were injected i.p. with 5-bromo-2 V-deoxyuridine (BrdU, 200 mg/kg; Sigma-Aldrich Kft, Budapest, Hungary). After 6 h, 5–7 μm thick tissue sections were excised from the newly frozen tumors and used for the detection of BrdU positive cells using an anti-BrdU monoclonal antibody (Becton Dickinson Hungary Kft., Budapest, Hungary), according to the manufacturer’s protocol. Positive cells were visualized with TRITC-conjugated anti-mouse IgG (1:100, Sigma). Endothelial cells were distinguished from tumor cells by treating the samples with rat anti-mouse CD31 antibody and labelled with biotinylated anti-rat IgG and streptavidin-FITC (Vector Laboratories, Burlingame, CA). The nuclei were stained with the Hoechst 33,342 dye (Molecular Probes, Eugene, OR). The number of labelled HT-29 tumor cells and the quantity of CD31-positive samples were determined in two different tumors by microscopic analysis of 6 independent regions. The vascularization extent of primary tumors was determined by microscopic analysis of two different living tumors, where four independent areas of living cells were counted and the percentage of separated endothelial cells covering a 10× magnification field of vision was calculated [12 (link)].