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How specific is your IHC antibody?

 

Validating an antibody for immunohistochemistry (IHC) use involves more than just observing a signal in cells. Cell Signaling Technology (CST) thoroughly tests the specificity of all antibodies recommended for IHC, and establishes optimal assay conditions to help you achieve the best results right from the start.

Verification of Specificity

  • Testing paraffin-embedded normal & diseased tissue of known target expression
  • Subjecting cells to phosphatase treatment to verify phospho-specific signal
  • Using xenografts and mouse models of cancer with known target expression
  • Using blocking peptides and knock out models
  • Confirming specificity using western blot

Verification of Phospho-specificity using λ-Phosphatase

Untreated

λ-Phosphatase-treated

IHC analysis of untreated and phosphatase-treated paraffin-embedded human lung carcinoma shows that the specific signal of phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb #8516 disappears after phosphatase treatment.

Verification of IHC Staining Specificity by Western Blot

CST Phospo-Met XP® #3077

Phospo-Met Competitor Ab

IHC analysis of phospho-Met in paraffin-embedded HCC827 xenograft shows staining for both products.

However, western blot analysis to verify specificity shows that the CST Phospho-Met (Tyr1243/1235) (D26) XP® Rabbit mAb #3077 is specific, while the competitor’s antibody cross-reacts with other phosphorylated tyrosine kinases.

 

Protocol Optimization

  • Optimal antibody dilution, antigen retrieval conditions, and protocols are predetermined and specified
  • Control slides available

Optimization of Detection Conditions

Biotin-based detection

SignalStain® Boost IHC

Analysis of paraffin-embedded human papillary renal cell carcinoma using Phospho-Met (Tyr1234/1235) (D26) XP® Rabbit mAb #3077 followed by biotin-based detection or SignalStain® Boost Detection Reagent (HRP, Rabbit) #8114. The use of the sensitive SignalStain® Boost Detection Reagent offers superior sensitivity and reduced biotin background staining.

 

 

 

Lot-to-lot Reproducibility

Comparison of Lot #9 with Lot #7

IHC titration analysis of adjacent sections of paraffin-embedded human colon carcinoma using Phospho-p44/42 MAPK (Erk 1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 shows the optimal antibody dilution remains 1:400 from lot to lot.

 

IHC-validated XP rabbit monoclonal antibodies from CST demonstrate the exceptional specificity and sensitivity required for the most accurate results in IHC and many other research applications. XP ‘eXceptional Perfomance’ monoclonal antibodies directed against novel targets provide new insights for basic and clinical researchers and scientists developing new drugs.

Phospho-Akt (Ser473) (D9E) Antibody Comparison

Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 was compared to three competitors’ IHC-approved Phospho-Akt (Ser473) antibodies.
(A) The optimal dilution of each antibody was individually evaluated to maximize specific signal in untreated LNCaP cells (left) and minimize non-specific signal in LNCaP cells treated with LY294002 (PI3 Kinase Inhibitor) #9901 (right).
(B) The determined optimal dilution for each antibody was used in IHC analysis of paraffin-embedded human breast carcinoma. Note stronger staining obtained with the XP® monoclonal antibody.


XP® Monoclonal Antibodies – Top Targets for IHC

Cat# Name Application Species
4060 Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb W IP IHC-P IHC-F IF-IC F H M R Hm Mk Dm Z B (C) (X) (Dg) (Pg)
8768 Stat3α (D1A5) XP® Rabbit mAb W IP IHC-P IF-IC ChIP H M R Hm Mk (Pg)
3777 Phospho-EGF Receptor (Tyr1068) (D7A5) XP® Rabbit mAb W IHC-P IF-IC F H M R Mk
4267

EGF Receptor (D38B1) XP® Rabbit mAb

W IP IHC-P IF-IC F H M Mk
4290 Her2/ErbB2 (D8F12) XP® Rabbit mAb W IHC-P H (M) (R)
4370 Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb W IP IHC-P IF-IC F H M R Hm Mk Mi Dm Z B Dg Pg Sc (C) (Ce)
3077 Phospho-Met (Tyr1234/1235) (D26) XP® Rabbit mAb W IP IHC-P IHC-F IF-IC F H M R
9188 PTEN (D4.3) XP® Rabbit mAb W IP IHC-P H M R Mk Dg (C)
12708 HER3/ErbB3 (D22C5) XP® Rabbit mAb

W IP IHC-P IF-IC F

H (M) (R) (Mk)
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