Purple Substrate as a DAB Alternative

January 06, 2016

University at Buffalo
Center for Hearing & Deafness
Principal Investigator

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Company:

Vector Labs

Product Name:

ImmPACT VIP

Catalog Number:

SK-4605

The goal of this experiment was to find an alternative to brown DAB staining for immunohistochemistry. We often work with tissues that have pigmentation and the brown stain of DAB can be difficult to distinguish from the brown pigment. We chose ImmPACT VIP for the purple color and hoped for compatibility with our existing HRP staining kit. This was a pilot experiment to see if ImmPACT VIP would work with our HRP staining kit.

Experimental Design and Results Summary

Application

Immunocytochemistry

Starting Material

Y79 human retinoblastoma cells

Protocol Overview

Y79 cells were fixed in cytospin solution and spun onto slides for staining. We permeabilized cells with PBS-Tween for 5 minutes and used CRF polyvalent HRP polymer kit (Scytek) in concert with our primary antibody (rabbit anti-caspase-3, Pharmingen 67341A). After blocking, the primary antibody was incubated for one hour at room temperature, rinsed in PBS, then polymer for one hour at room temperature. After 3 rinses in PBS, ImmPACT VIP substrate was incubated for 5 minutes. Slides were coverslipped and evaluated for staining results by standard brightfield microscopy.

Tips

We tried a few different incubation times for the ImmPACT VIP substrate and five minutes was optimal for our purposes.

Results Summary

The purple substrate worked very well without any background problems on the first try. The results showed us a very intense, but specific, purple color that convinced us that the substrate will be appropriate for our needs.

Additional Notes

You may want to optimize incubation time between 2-12 minutes .

Image Gallery

Summary

The Good

Very bright, purple and specific

The Bad

None noted

The Bottom Line

This is a very useful alternative to brown DAB staining, especially in tissues containing pigment. The substrate was compatible with our standard HRP kit. This product is also advertised for use in double-staining with other substrate colors available.

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