Biotin-tyramide

Biotin-tyramide for electron microscopic immunocytochemistry^[1]

This experimental protocol is organized based on research content and is for reference only. Please adjust according to actual needs.

(1) Reagent and antibody preparation: 1% glutaraldehyde fixative prepared in 0.1M phosphate buffer (store at 4°C); 1% ovalbumin blocking solution prepared in 10mM PBS (pH=7.2); primary antibody against target protein; corresponding secondary antibody; streptavidin-HRP diluted 1:500 with PBS; Biotin-tyramide diluted 1:50 with 1× amplification diluent, prepare immediately before use, diluted solution should not be stored for more than 2 weeks.

(2) Tissue processing and embedding: Tissue pieces immersed in 1% glutaraldehyde in 0.1M phosphate buffer at 4°C for 2h, washed with phosphate buffer, then embedded in Lowicryl at low temperature according to standard procedures.

(3) Sectioning and grid mounting: Ultrathin sections cut and mounted on carbon coated nickel grids for use.

(4) Immunolabeling and Biotin-tyramide deposition: Grids floated face-down on 1% ovalbumin (in PBS, pH=7.2) at room temperature for 30min; transferred to corresponding primary antibody at room temperature for 2h or at 4°C overnight; rinsed with PBS for 15min, 1% ovalbumin for 15min, transferred to corresponding biotinylated secondary antibody at room temperature for 60min; rinsed with PBS for 15min, 1% ovalbumin for 15min, transferred to streptavidin-HRP 1:500 at room temperature for 30min; rinsed with PBS for 15min, transferred to Biotin-tyramide 1:50 (in 1× amplification diluent) at room temperature for 10min; after rinsing with PBS and distilled water, stained with uranyl acetate and observed under electron microscope.

Precautions:

(1) The 1% ovalbumin blocking step should not be placed before Biotin-tyramide incubation, otherwise the HRP-tyramide reaction will be interfered.

(2) Biotin-tyramide dilution should be freshly prepared; storage for more than 2 weeks will cause significant decrease in labeling intensity.

(3) For safety and health, please wear lab coat and disposable gloves, and handle glutaraldehyde and uranyl acetate in a fume hood.

References:
[1] Mayer G, Bendayan M. Biotinyl-tyramide: a novel approach for electron microscopic immunocytochemistry. J Histochem Cytochem. 1997 Nov;45(11):1449-54.