Scytek HRP Polymer Kit for Immunostaining

January 15, 2016

University at Buffalo
Center for Hearing & Deafness
Principal Investigator

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Company:

Scytek

Product Name:

CRF Anti-Polyvalent HRP Polymer (DAB) Stain Kit

Catalog Number:

CPH-080

We are studying Her2 expression in a number of cancer cell lines by immunocytochemistry. We selected this HRP polymer kit because our previous kit had been discontinued and we needed to find a new product for our routine staining experiments. This kit was appealing because it contained blocking solutions and is compatible with mouse, rat, rabbit and guinea pig primary antibodies.

Experimental Design and Results Summary

Application

Immunocytochemistry

Starting Material

BT474 breast cancer cells

Protocol Overview

BT474 breast cancer cells were fixed in cytospin solution and spun onto slides. Cells were permeabilized with PBS-Tween, then blocked with the following Scytek kit components: H2O2 Block (5 minutes) and Super Block (4 minutes). Cells were incubated in rabbit anti-HER2 primary antibody (Genetex) for one hour, followed by three rinses in PBS. CRF Polymer from the kit was incubated for 30 minutes, followed by 3 rinses in PBS and one rinse in water. The DAB substrate was prepared freshly, as instructed by the kit and incubated for 5 minutes (less than recommended by the kit).

Tips

The instructions given are for paraffin sections, so for cytospins, you may need to decrease the incubation time as we did for the DAB step.

Results Summary

We saw a nice reddish-brown DAB stain in the BT474 cells, which were our positive control for HER2. The negative control was very clean.

Additional Notes

None

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Summary

The Good

The kit is self-contained with everything you need for blocking, detection and DAB reaction.

The Bad

You may need to adjust incubation times, depending on your sample. But this is very minor.

The Bottom Line

In summary, we were very pleased with the performance of this kit right away. The negative controls have virtually no background, while the positive control shows the expected brown staining.

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