Streptavidin biotin blocking kit

Immunohistochemical analysis was performed on 10 μm fresh-frozen sections of P19 placental tissue blocks (n = 3 animals). Frozen sections were fixed with 4% paraformaldehyde/PBS, and endogenous peroxidase was quenched by immersing in 0.3% (v/v) hydrogen peroxide/methanol. A streptavidin/biotin blocking kit (Vector Laboratories, Burlingame, CA) was used to block endogenous biotin according to the manufacturer's instructions. After 30 min of incubation with 10% normal goat serum, the sections were incubated at 4°C overnight with a rabbit anti-mouse CD11b polyclonal antibody (1:100 dilution, 0.5 mg/ml, ab75476, Abcam), a rabbit anti-mouse trophoblast specific protein alpha (TPBPA) antibody (1:100 dilution, 1 mg/ml, ab104401, Abcam), or a normal rabbit IgG (1:40 dilution, 0.4 mg/ml, sc-2027, Santa Cruz Biotechnology, Inc., Dallas, TX) as a negative control. Subsequently, the sections were incubated at room temperature for 1 h with a goat anti-rabbit IgG biotin conjugate (1:800 dilution, B8895, Sigma–Aldrich, St. Louis, MO). The immunoreactivity was visualized using the avidin-peroxidase (1:400 dilution, E2886, Sigma-Aldrich) and AEC substrate kit (Thermo Fisher Scientific, Inc., Waltham, MA) according to the manufacturer's instructions and then examined under light microscope (BX-51, Olympus, Tokyo, Japan).

Chicken and duck muscle cells were grown on 24-well Nunclon flat-bottom plates (Nunc), as they did not proliferate well on glass coverslips. Lectin labeling was performed as previously described (40 (link)). Cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Endogenous biotin activity was blocked by the use of a streptavidin/biotin blocking kit (Vector Laboratories). Cells were then incubated overnight at 4°C in the dark with fluorescein isothiocyanate (FITC)-labeled Sambucus nigra agglutinin (SNA) lectin (human α-2,6-linked sialic acid receptor binding) and biotinylated Maackia amurensis agglutinin II (MAA II) lectin (avian α-2,3-linked sialic acid receptor binding) (Vector Laboratories), both at 10 μg/ml. Subsequently, the cells were washed three times with TBS and incubated for 2 h at room temperature with a streptavidin-Alexa Fluor 594 conjugate (Invitrogen). Cells were washed again three times with TBS and mounted with ProLong Gold antifade reagent with 4′,6′ diamidino-2-phenylindole (DAPI) (Invitrogen). Negative controls were processed without the use of lectin.