Double Immunofluorescence Staning

November 24, 2015

Texas Tech University Health Science Center
Internal Medicine
Postdoctoral Researcher

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Company:

Invitrogen

Product Name:

Hoechst 33342

Catalog Number:

H3570

Hoechst is a nucleic acid stain which facilitates cell permeant nuclear counterstain and emits blue fluorescence when bound to the dsDNA.

Experimental Design and Results Summary

Application

Double Immunofluorescent Staining

Starting Material

Tricuspid Valve Cross Sections

Protocol Overview

Fix the sections with 50% Methanol and 50% Acetone for 15 min. Wash trice with PBS for 5min.Add 0.5% triton X-100 and incubate for again 15 min. Wash with PBS again twice and Block with 5%BSA + 5% Goat serum and block it for 30 min.Then add primary antibody 1:200 in 3%BSA and incubate it for over night. After incubation wash again with PBS and add secondary antibody 1:500 in 3%BSA and incubate it for 1hr.Prepare hoeschst in 1:1000 in PBS and incubate it for 5 min wash with PBS and use prolong mounting medium and cover it with cover slip.

Tips

In initial experiments it is better to try several concentrations and dtermined the suitable concentration for optimal staining.

Results Summary

Hoeschst is a fluorescence dye which binds to dsDNA at stretches of at least three AT base pairs, but does not bind to stretches of two or more GC base pairs. It is mainly used to stain live cells.

Additional Notes

None

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Summary

The Good

It works great.

The Bad

None

The Bottom Line

Hoechst is a cell permeable nucleic acid dye. Due to its fluorescence it brings conformational changes in the chromatin of the cells. Subsequently they can detect gradations of nuclear damage. It binds specificaly to the AT rich dsDNA strands and enhance the fluorescence than the GC rich bands. It plays a major role in double immunofluorescent staining.

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