HER2/Neu mAb from Santa Cruz for Paraffin IHC

November 21, 2016

Center for Hearing & Deafness
University at Buffalo
Principal Investigator

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Company:

Santa Cruz Biotechnology

Product Name:

Neu (3B5)

Catalog Number:

sc-33684

We need HER2 antibodies for a collection of paraffin-embedded tumor sections for our studies on HER2 expression in retinoblastoma. For this application, we need the antibody to work on paraffin sections. This HER2/Neu antibody from Santa Cruz is a mouse monoclonal that can pair with rabbit primary antibodies for double staining.

Experimental Design and Results Summary

Applications

Immunohistochemistry

Sample

5 micron paraffin section of a xenograft of human N87 cells (gastric cancer met)

Primary Incubation

2ug/ml of Neu (3B5): sc-33684 in PBS-Tween buffer at room temperature for one hour.

Blocking Agent

None

Secondary Incubation

Sigma goat anti-mouse FITC conjugate, 1:300 at room temperature for 45 minutes

Tertiary Incubation

Vectashield mounting medium containing DAPI counterstain

Detection

Fluorescence microscopy- Nikon microscope with SPOT camera

Results Summary

The paraffin section of the N87 xenograft was rehydrated through a series of xylenes and alcohols, boiled in sodium citrate buffer pH 6.0 and permeabilized with PBS-Tween at room temperature for 10 minutes. Antibody staining was carried out as described above. As seen in the attached photo, the HER2 staining was membrane-associated and very intense green (FITC).

DOI/PMID#

N/A

Additional Notes

We use 2 ug/ml for both paraffin sections and immunocytochemistry.

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Summary

The Good

Very specific with low background, even in a mouse xenograft

The Bad

None noted

The Bottom Line

We have found this antibody to be very useful for both paraffin sections and cytospins containing HER2+ cell lines. Since it is a mouse monoclonal, it can be used in combination with rabbit, sheep, goat or other compatible primary antibodies for dual and triple stains.

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