Principle:

When cells are incubated with a primary antibody, The primary antibody recognizes and binds to cell surface antigens but since there is no way to detect it’s presence on the cell surface, the cells are subsequently incubated with a secondary antibody.The secondary antibody can be conjugated to a fluorochrome or biotin that can then be detected in several different ways. In addition to these conventional immunostainings, a newer method has been introduced with increased sensitivity. This is called the tyramide signal amplification (TSA) method. The secondary antibody recognizes the primary antibody bound to concerning antigen and binds to it and in that way provide a reporter for the primary antibodies bound to the cell surface.

Solutions required for Antibody staining:

1 Fixative : Formaldehyde ( 4- 10% depending on tissue to be fixed),PBS,Water.(Formaldehyde solutions are used as a fixative for microscopy and histology.)

2. PBT BS (Final 1X), TWEEN 20 for embryos / Triton X for imaginal discs ( conc 0.1 % final),Water.

A detergent like triton or tween is used to permeabilize eukaryotic cell membranes.

Protocol:

Rinse the fixed embryos with absolute ethanol and then with methanol twice.

Rinse with PBT twice

Block the embryos in PBT with 1% BSA(Bovine serum albumin) for 1 hr.(Albumin is used to solubilize lipids and it also increase the affinity)

Add primary antibody and incubate Over night at 4°c.

Remove antibody and wash with PBT for 1 hr.

Add secondary antibody and incubate for 1 hr.

Remove and wash with PBT by intermittently changing the solutions.

Mount the embryos and observe in Uv.

Trouble shooting:

If the primary antibody renders weak signal or no signal,one can try to preabsorb the antibody or for weak signal TSA kit works very well.

Tyramide Signal Amplification is a technology that amplifies both chromogenic and fluorescent signals in standard immunohistochemistry protocols. The result is a significant increase in sensitivity.

Tyramide is the amplification reagent. It is a phenolic compound that, when activated by the enzyme horseradish peroxidase (HRP), covalently binds to electron rich moieties on a surface (i.e., predominantly to tyrosine residues in proteins in tissue or cell preparations)

I have used this kit and believe me it does wonder,even for antibodies which never gave any signal with flourochrome dyes.

Sometimes situation demands you to use two antibodies raised in same organism which normally shows cross reaction when used for double staining,but this can reduced or minimised completely by refixing the tissue (embryo or imaginal discs ) after immuno staining with one antibody and then proceed with another.Most of the time this trick works.

Example of antibody staining in wing imaginal dics

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