FFPE Immunohistochemistry of C5b-9

December 02, 2014

Duke University Medical Center
Associate Professor

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Company:

Abcam

Product Name:

Anti-C5b-9 antibody [aE11]

Catalog Number:

ab66768

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Browse Similar Products: C5b-9 Antibodies

Activation of the complement system ultimately leads to the formation of the membrane attack complex, or MAC. The MAC is composed of the complement components C5b, C6, C7, C8, and multiple copies of C9. This complex is frequently referred to as C5b-9. In the context of the C5b-9 complex, a cryptic epitope is exposed in C9. The aE11 antibody recognizes this epitope and can, therefore, be used to identify the MAC in cell membranes or in blood. In the current study, we used the aE11 antibody to stain FFPE sections of kidney from a patient with the autoimmune disease systemic lupus erythematosus (SLE).

Experimental Design and Results Summary

Applications

Immunohistochemistry

Sample

FFPE sections of SLE kidney

Primary Incubation

The aE11 antibody was diluted 1:100 (final concentration 10 µg/ml) and incubated on the slide for 1 h at room temperature (RT).

Blocking Agent

After Pronase (BioGenex) digestion for 10 minutes, the slide was subjected to peroxide block for 5 minutes, followed by protein/serum block for 10 minutes. The protein/serum blocking solution is made in the lab and consists of avidin/biotin blocking reagents, normal sera from multiple species, and a blocking reagent from BioCare. All incubations were at room temperature.

Secondary Incubation

Vector anti-mouse ImmPress secondary reagent for 30 minutes at room temperature.

Tertiary Incubation

NA

Detection

Vector DAB for 2 minutes at room temperature.  The slide was then exposed to Vector Hematoxylin for 30 seconds at room temperature followed by standard dehydration, clearing, and mounting.

Results Summary

The stain shows excellent specific staining of glomeruli and in the region of peritubular basement membranes. At the dilution used (1:100) there was very little background or non-specific staining.

Additional Notes

None

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Summary

The Good

Strong specific staining of the expected structures with minimal optimization.

The Bad

Nothing bad to report. The antibody performed as advertised.

The Bottom Line

This is an excellent product that performs exactly as one would hope. Coupled with Abcam's money-back guarantee, you really can't go wrong.

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