To reduce non-specific binding, slides were incubated in 1 % BSA in PBS for 30 minutes.
To reduce non-specific binding, slides were incubated in normal goat serum (Biospa, Milan, Italy) for 10 min at room temperature, before overnight incubation with primary Ab in a humified chamber at 4°C.
To reduce nonspecific antibody binding, slides were incubated in 1% bovine serum albumin in PBS for 1 h at room temperature before incubation with rabbit polyclonal antibody to human AIF overnight at 4 °C.
Midguts and washed salivary glands were placed in separate drops of PBS on silane-treated glass slides (BioWorld, Dublin, OH), dried, and fixed with acetone for 10 min. To block nonspecific binding the slides were incubated with 10% bovine serum albumin (BSA) in PBS at room temperature for 30 min, and then stained with fluorescein isothiocyanate (FITC -conjugated rabbit anti-B.
To block aspecific binding the slides were incubated with Tris-buffered saline (TBS) supplemented with 5% goat serum.
Endogenous peroxidase activity was blocked with 3%H2O22 in methanol for 10 min. After pretreatment with Tris-EDTA buffer, in order to block non-specific antibody binding, the slides were incubated with 10% goat serum in PBS for 30 min. Primary monoclonal ERα antibody (ER-6F11, Novocastra, Newcastle, UK) was used at 1 50 dilution and detected following standard immunohistochemical techniques.
After nonspecific binding was blocked, the slides were incubated with NDRG2 antibody (1 200; Abnova, Taipei, Taiwan) or GLUT1 antibody (1 200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in phosphate-buffered saline (PBS) at 4°C overnight in a humidified container.
In order to block the nonspecific binding of the antibody, the slides were incubated for 30 min with 5% bovine seroalbumin (BSA) in PBS at room temperature.
After blockage of non-specific binding sites with 10% goat serum, slides were incubated with anti-ZO-1 for 1 h.
To block nonspecific binding of the secondary antibodies, the slides were incubated with 4% donkey serum diluted in blocking buffer for 30 min.
After non-specific binding was blocked by rabbit serum, the slides were incubated with the culture supernatant of the hybridoma producing the anti-H3.5 monoclonal antibody (1 100 dilution) for 24 h at room temperature, and endogenous H3.5 was visualized using the avidin biotin complex method.
