Antibody Validation for Immunohistochemistry
Immunohistochemistry (IHC) involves the labeling of cellular proteins in cell pellets or tissues with a specific primary antibody and visualization of the target with a IHC detections reagent. Cell Signaling Technology offers over 500 antibodies validated for IHC, as well as IHC diluents, detection reagents, and controls, to ensure that your IHC studies yield accurate results. The determination of target specificity in immunohistochemical analysis requires multiple validation steps. Scientists at Cell Signaling Technology use a variety of approaches for each antibody validated for IHC to demonstrate that staining achieved with the antibody is specific and believable.
Validation Steps Include
- Western blot analysis is performed to demonstrate specific bands of the appropriate molecular weight(s), with minimal cross-reacting bands.
- Paraffin-embedded cell pellets of known target expression levels are used to verify target specificity.
- Antibody performance is assessed in relevant mouse models of cancer.
- Xenografts generated from cell lines with known target expression levels help verify target specificity.
- Human cancer tissue arrays are used to demonstrate antibody performance over a broad spectrum of tissue types.
- Staining on fresh frozen tissues is performed when appropriate.
- Tissue sections and cell pellets are subjected to phosphatase treatment to verify target phospho-specificity.
- The use of blocking peptides verifies specificity and rules out Fc-mediated binding, biotin background, and other non-specific staining.
- Thorough lot testing ensures the reproducibility necessary for accurate IHC results.
- Dilutions and protocols are predetermined and specified; control reagents are also available.
Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060, Akt (pan) (C67E7) Rabbit mAb #4691: IHC analysis of SignalSlide® Phospho-Akt (Ser473) IHC Controls #8101 using #4060 (upper) or #4691 (lower).
Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060: IHC analysis of paraffin-embedded WT (left) and PTEN (-/-) (right) mouse prostate using #4060. Tissue courtesy of Dr. David Guertin, The Whitehead Institute for Biomedical Research, Cambridge, MA.
Xenograft and Phosphatase Treatment
Phospho-EGF Receptor (Tyr1068) (D7A5) XP® Rabbit mAb #3777: IHC analysis of paraffin-embedded HCC827 xenograft, control (upper) or λ phosphatase-treated (lower), using #3777.