And, the successful binding of avidin-biotin conjugate was tested using the fluorescein-conjugated antibiotin
antibodies under the visualization of fluorescence microscope.
horseradish peroxidase (HRP) will generate a tyramide radical that cross-links a biotin or a fluorophore to all exposed tyrosine residues of any protein near the recognition event (6).
Slides were then washed twice with 4x SSC-0.2% SDS buffer and 2x SSC buffer, and hybridization was detected by the sequential addition of Cy5-conjugated mouse antibiotin
immunoglobulin G (IgG) (Jackson ImmunoResearch, West Grove, PA, USA) and Cy5-conjugated goat antimouse IgG (Jackson ImmunoResearch).
Following hybridization, slides were incubated successively with antibiotin
antibody and fluorescein-conjugated IgG, counter-stained with propidium iodide and mounted in Vectashield medium (Vector Laboratories).
Peritoneal B-1a cells were isolated using magnetic microbeads conjugated to a cocktail of biotinconjugated antibodies against non-B-1a cells as first labeling reagent (B-1a Cell Biotin-Antibody Cocktail) and AntiBiotin
MicroBeads as secondary labeling reagent (Miltenyi Biotec), following the manufacturer's instructions.
The PBMCs were magnetically labelled with a cocktail of biotin-conjugated antibodies against CD3, CD7, CD16, CD19, CD56, CD123, and Glyocophorin A and antibiotin