Rabbit UltraAvidin DyLight® 550 Staining Kit

The immunofluorescent staining method is preferred to distinguish fine subcellular structures in fixed tissue sections. The avidin-biotin system for immunostaining has increased the sensitivity for detecting very small amounts of antigen and definitive negative controls can be used as compared to the direct method of labelling.1-3 In this method, the secondary antibody directed against the immunoglobulin of the species in which the first antibody is raised, is biotin labeled. This is allowed to react with the primary antibody, followed by the addition of the DyLight 550-conjugated UltraAvidin forming a complex whereby many DyLight 550 molecules are indirectly bound to each secondary antibody.4,5 The avidin has an extraordinary affinity for the small molecule biotin forming a noncovalent bond with a dissociation constant of 10-15M making this system highly stable.5,6 The bound conjugate is then visualized using a fluorescence microscope. The fluorochrome is excited by application of an appropriate light source, the light emitted being filtered by appropriate filters to remove the excitation wavelength and then led to the observer's eye. DyLight 550 has a maximum absorbance at 550 (green) and an emission maximum at 576nm (red-orange). It is covalently attached to UltraAvidin and purified to assure an optimal fluorochrome/protein molar ratio.

Leinco's UltraAvidin is a unique form of avidin, modified to prevent non-specific binding on cell surfaces either due to lectin-like receptor adherence or electrostatic interactions. The secondary Goat anti-rabbit IgG antibody is a F(ab/)2 fragment, eliminating non-specific Fc receptor binding and is especially suitable for immunohistochemical studies. It is affinity isolated using rabbit IgG and is adsorbed against human serum proteins and mouse IgG to reduce cross-reactivity with human or mouse tissue sections. The antibody is then conjugated to biotin at a molar ratio to optimize performance. The kit has been developed for enhanced sensitivity whereby low amounts of antigen may be detected or where lower concentrations of primary antibody may be used.

Materials Supplied
1. UltraBlockTM Blocking Buffer (Leinco Part No.: B395): 4ml
2. Reagent A: Goat Anti-Rabbit IgG-Biotin (Leinco Part No.: R106): 1ml
3. Reagent B: UltraAvidin DyLight 550 (Leinco Part No.: A426): 1ml
4. Mixing Bottles with drop dispenser: NOTE: These are used to prepare the working solutions for the staining kit and to dispense the required amount on each tissue section. (Note: If held vertically upside down the volume of each drop is 45-50 ul. Holding the bottle at an angle would increase drop volume.)

Note: Keep bottles closed to prevent microbial or dust contamination. Do not reuse working solutions.

Materials Required But Not Supplied
1. Cryostat or paraffin tissue sections.
2. Xylene
3. Ethanol
4. Suitable diluting buffer such as 0.01M Phosphate buffered saline, pH 7.2 (PBS) (Leinco Part No.: P364).
5. Primary Antibody
6. Counterstain
7. Mounting Medium (Mix 1 part glycerol with 2 parts PBS).

Working Solutions Required:
1. Blocking Buffer: Add 3 drops of Blocking Buffer from the Rabbit UltraAvidin DyLight 550 Staining Kit to 10ml PBS in the Mixing Bottle 1. Snap on drop dispenser tip.
2. Primary Antibody: Adjust to required concentration with PBS.
3. Biotinylated secondary antibody: Add 1 drop of Reagent A from the Rabbit UltraAvidin DyLight 550 Staining Kit to 10ml PBS in Mixing Bottle 2.
4. UltraAvidin-DyLight 550: Add 50ul from the Reagent B of the Rabbit UltraAvidin DyLight 550 Staining Kit to 10ml PBS in Mixing Bottle 3.

Suggested Method All the steps are carried out at room temperature in moist chambers.

1. For cryostat sections fix in acetone for 10 mins and air dry. Go directly to Step 5.
2. For paraffin sections fix at 35C for 2-3 minutes.
3. Deparaffinize in xylene for 20 mins.
4. Wash in decreasing grades of ethanol, 10 mins. in each.
5. Wash under running tap water for 10 mins.
6. Wash in PBS for 10 mins.
7. Wipe slide and add the blocking serum dropwise onto the slide, covering the section. Incubate for 20 mins.
8. Drain out blocking buffer and add primary antibody onto the section, covering it. Incubate for 1 hour.
9. Drain out antibody and wash in PBS for 10 mins.
10. Wipe slides and add the biotinylated secondary antibody onto the section, covering it. Incubate for 30 mins.
11. Drain out antibody and wash in PBS for 10 mins.
12. Wipe the slides and add the UltraAvidin-Rhodamine onto the section, covering it. Incubate for 30 mins.
13. Drain out solution and wash in PBS for 10 mins.
14. Counterstain and mount in glycerol.

(Note: Fluorochrome staining is not stable and cannot be preserved. For best results, it should be observed and photographed, if needed, immediately after processing the section.)

These instructions are for the use of Leinco's Rabbit UltraAvidin DyLight 550 Staining Kit for immunohistology. The rabbit primary antiserum/ antibody is supplied by the user. Reagents supplied are sufficient for at least 1000 tissue sections. When not in use the reagents should be stored at 2-8^C.

1. Peter, J.H. and Baumgarten, H (Eds.) (1992) Monoclonal Antibodies Springer-Verlag, New York Berlin Heidelberg
2. Green, M. A.. et al. (1989) J. Clin. Pathol. 42:875-880
3. Giorno, R. (1989) Diag. Immunol. 2:161-166
4. Heyderman, E. (1979) J. Clin. Pathol. 32:971-978
5. Guesdon, J-L; et al. (1979) J. Histochem. Cytochem. Vol.27, No.8:1131-1139
6. Hsu, S-M; et al. (1980) Am. J. Clin. Pathol. Vol. 75, No.5:734-738
7. Young, P. R. (1989) J. Virol. Methods 24:227-236