Immunohistochemistry (IHC) – Direct labeling

Lightning-Link® antibody labeling kits can be used to directly conjugate antibodies for IHC to HRP or other labels. The IHC data below shows that it is possible to obtain very good staining whilst avoiding the use of secondary antibodies, therefore saving significantly in time and reagent costs.

Anti-human CD20 (clone L26) was conjugated to HRP using Lightning-Link® kits, and used in IHC in comparison to a traditional indirect technique

IHC - Comparison of anti-human CD20 (clone L26) conjugated to HRP using Lightning-Link® kit and a traditional indirect technique

This shows a comparison of the anti-CD20 antibody L26 labelling paraffin-embedded bone marrow, both in a standard indirect technique with Dako Envision as the detection system (used as a control) and the same antibody directly labelled with HRP using Lightning-Link®. There are examples of the antibody at different concentrations, and with different HRP labelling ratios – all show clear staining of the B lymphocytes, without background staining.

Dual staining of CD3 and CD20 with directly conjugated antibodies using Lightning-Link® kits


IHC staining using lightning-link HRP and AP


Brown = CD20 labeled with HRP

              Red = CD3 labeled with Alkaline Phosphatase

Mouse anti-human CD20 was conjugated to HRP using Lightning Link® kits, and rat anti human CD3 was conjugated to Alkaline Phosphatase using Lightning Link® kits.   Both antibodies were then used simultaneously to staining formalin fixed paraffin embedded sections of human lymph node. CD20 positive cells are visualised with DAB and are stained brown, and CD3 visualised with Fast Red and stained Red.

Direct labelling of primary antibodies with Lightning-Link® kits enables very simple dual colour staining in IHC, which is otherwise a highly complex technique.

View the Lightning-Link® HRP kits.

View the Lightning-Link® Alkaline Phosphatase kits.

The following publications utilised Lightning-Link® kits as part of their IHC methodology:

  1. Stromal Cell-Derived Factor-1alpha Activation of Tissue-Engineered Endothelial Progenitor Cell Matrix Enhances Ventricular Function After Myocardial Infarction by Inducing Neovasculogenesis
  2. Human Mesenchymal Stem Cells Self-Renew and Differentiate According to a Deterministic Hierarchy
  3. Restricted access of proteins to mannan polysaccharides in intact plant cell walls
  4. TCF7L2 rs7903146 impairs islet function and morphology in non-diabetic individuals