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For experiments, freshly seeded SCs were allowed to attach O.N. in regular media, changed to DMEM containing 10% of newborn bovine serum (NBS) depleted of retinoids (achieved by sequential incubations with AG1-X8 resin and charcoal), in the presence of 2 µM forskolin and 60 µg/ml BPE.
This was then followed by sequential incubations with blocking solutions containing primary and secondary antibodies.
A12 was detected by sequential incubations with the mouse anti-VSV-G P5D4, biotinylated anti-mouse IgG (Vector, Burlingame, CA), and avidin-biotin peroxidase complex (Vector).
Primary antibody binding was detected by sequential incubations with horseradish peroxidase (HRP -labeled appropriate secondary and tertiary antibodies (obtained from DAKO, Glostrup, Denmark).
After washing with Tris buffered saline (TBS) with tween, the endogenous biotin was suppressed by sequential incubations with 0,1 % avidin and 0,01 % biotin (Dako, Glostrup, Denmark) for 10 min each at room temperature.
Alternatively, the cells were blocked in goat γ-globulin (200 μg/ml; Jackson Immunoresearch) before the detection of E-cadherin by sequential incubations with monoclonal anti-E-cadherin (1 50 dilution; BD Bioscience), followed by biotinylated goat anti-mouse antibody (5 μg/ml; Jackson Immunoresearch), and finally by Alexa-streptavidin (1.2 μg/ml; Molecular Probes).
After hybridization, lacZ mRNA was visualized by sequential incubation with Rabbit Anti-fluorescein (1µg/ml final) and Alexa Fluor 647 Donkey Anti-rabbit (1µg/ml) antibodies (Molecular Probes).
Immunoreaction for the HPV16-L1 was detected by sequential incubation with biotinylated goat anti-mouse immunoglobulin secondary antibody (1∶200 from Vector Laboratories, Burlingame, CA) and streptavidin Texas red (1∶400, Vector Laboratories, Burlingame, CA) both for 60 minutes at room temperature.
Immunoreaction was detected by sequential incubation with biotinylated goat anti-mouse immunoglobulin secondary antibody (1∶200, Vector Laboratories, Burlingame, CA) and streptavidin Texas red (1∶400, Vector Laboratories, Burlingame, CA) both for 60 minutes at room temperature.
Sections were incubated with rabbit polyclonal anti-galanin antibodies (1∶100; Biozol, Eching, Germany ) or rabbit polyclonal anti-GLP1 antibodies (1∶100; Biozol, Eching, Germany) for 3 days at 4°C, followed by sequential incubation with biotinylated anti-rabbit IgG (Vector Labs., Burlingame, CA, USA) for 1 h and with ABC complex for 1 h at room temperature.
