The advantages of staining specimens

8th June 2015

A look at developments in multicolour enzymatic immunohistochemistry assays for formalin-fixed paraffin-embedded tissue

Immunofluorescence (IF) has become a preferred method of concurrently detecting multiple antigenic markers within a single tissue specimen. However, immunoenzymatic chromogen staining with multiple coloured substrates remains an informative and important research tool.

Staining specimens with immunoenzymatic chromogens allows researchers to cast a broader net for investigating targets because, unlike IF, it is permanent and can be visualised in relation to the comprehensive morphology of tissue specimens. This stability also allows standard histological stains to be used in conjunction with the immunohistochemistry (IHC) to give researchers an additional layer of information.

Immunofluorescence is often preferred over enzymatic IHC because it is a technically simpler method of visualising multiple antigenic markers.

Imaging with a fluorescent microscope and creating the composite images of multiple IF colour channels can be the most complicated aspect of IF staining, but quantification of distinctly stained elements is simple and precise.

The development of assays involving multiple IHC chromogenic substrates presents many challenges, such as determining the appropriate sequence of marker application/detection, compatibility of cellular localisation of combined markers, special requirements for preparation of various enzymatic substrates, visual contrast compatibility of chromogenic substrates, the length of the overall staining process, and methods of analysing staining results.

These potential IHC development obstacles can take time to overcome, but when the IHC assay is complete, the various chromogens can be visualised simultaneously, using standard light microscopy, and can be viewed repeatedly without altering staining results. These qualities of multicolour IHC are of significant value to researchers, especially in the early phases of study.

There are now many tools available to easily resolve some of the significant assay development obstacles of multicolour enzymatic immunohistochemistry. Abcam has developed kits for easy antibody conjugation (both Horseradish Peroxidase and Alkaline Phosphatase), and a range of chromogenic substrates with improved stability.

These products reduce the challenges of involved substrate preparation, and significantly reduce the length of staining procedures. The present work discusses how improved reagents simplify multicolour enzymatic IHC assay development for Formalin-Fixed Paraffin-Embedded (FFPE) tissues.

Materials and methods

To test a range of Abcam’s improved staining reagents, an experiment involving two immunohistochemistry markers (Active Caspase 3 and CD68) and one standard histology stain (Iron/Prussian Blue) was chosen. Two different IHC detection systems (Horseradish Peroxidase and Alkaline Phosphatase) were used to detect the two selected markers, in order to avoid reagent cross-reactions.

Chromogens with the highest available visual contrast were chosen to allow ease of analysis of staining results. This also required consideration of the colour product of the standard iron stain reaction (blue) and naturally occurring pigments in the tissues (brown). 

All tests were performed on 5μm thick sections of selected FFPE (immersion fixed in 10% neutral buffered formalin) human tissues (liver, spleen, tonsil).

Leica Bond Max Automated Stainer Procedures: All staining procedures were performed on the Bond Max automated stainer in order to generate the most standardised and reproducible results possible. Bond Dewax Solution was used to de-paraffinise FFPE sections. Bond Wash Buffer, equivalent to Tris Buffered Saline, was used as standard IHC wash buffer. Bond Peroxidase Block was used to inactivate endogenous peroxidases in all tissues. Heat-induced epitope retrieval was performed on the tissues at approximately 100°C using Bond Epitope Retrieval Buffers, equivalent to Citrate Buffer (~pH 6) and EDTA Buffer (~pH 8), for 10-20 minutes prior to incubation with IHC primary antibodies.

Double IHC Staining (with Abcam and Leica Bond detection reagents) + standard Iron Stain (with Abcam Iron Stain kit): Slides were placed on the Bond Max autostainer for staining with the Abcam antibody for Active Caspase 3 (Rabbit polyclonal, ab13847). 

The Active Caspase 3 antibody was conjugated to HRP complex using an Abcam HRP Conjugation Kit (ab102890), diluted to approximately 3μg/mL, applied to tissues for 15 minutes, and detected with Abcam Steady DAB/Plus (ab103723) for 5 minutes.

The slides were completely air-dried following DAB staining to prevent non-specific DAB-Iron reagent interaction, then rehydrated in deionised water for staining with the Abcam Iron Stain kit (ab150674) for two-three minutes.

Slides were thoroughly rinsed in deionised water and immediately placed back onto the Bond Max autostainer for staining with the Dako CD68 antibody (Mouse monoclonal [KP1], M 0814) diluted to approximately 0.032μg/mL, applied to tissues for 15 minutes, and detected by alkaline phosphatase staining with the Bond Polymer Refine Red Detection Kit (DS9390) and Abcam StayRed/AP Plus (ab176914) substituted for the Bond kit chromogen.

All slides were air-dried after staining procedures were completed, and coverslipped with Vector VectaMount permanent mounting medium (H-5000).

Results and discussion

To fully determine the optimal order of staining/detection for this staining combination, preliminary work was done to determine which of the IHC antibodies worked best with the available detection systems/chromogens. Testing showed that the harsh constituents of the iron stain working solution reduced the staining intensity of the IHC chromogens when used subsequently. This was more apparent with the alkaline phosphatase (AP) chromogen than with the horseradish peroxidase (HRP) chromogen, so the marker with the lowest apparent avidity was conjugated to the more robust HRP complex and diaminobenzidine (DAB) chromogen in order to best preserve its signal throughout the process. 

A non-specific staining interaction between the DAB chromogen and the iron stain reaction product was observed when iron staining was done first. Therefore, the optimal order for this staining combination was: Active Caspase 3 (Steady DAB/Plus), Iron Stain, CD68 (StayRed/AP Plus). Considerable testing was performed to attain the optimal intensity of each chromogenic stain in order to achieve an acceptable visual combination).

With Abcam’s new staining products, we were able to decrease the number of reagents involved in the multicolour stain assay, significantly reduce the assay run time, and effectively combine multicolour enzymatic chromogens with a standard histology stain.

The primary antibody HRP conjugation kit allowed for direct marker-chromogen detection, eliminating the need for indirect complex reagents and reducing assay run time by approximately one hour from that of our standard indirect detection methods.

Abcam’s new chromogens were comparable to those we currently use in terms of preparation and staining results, however, the Steady DAB/Plus reagent has a significantly longer period of stability compared with our other individual chromogens and allowed for simplified set-up when used on our standard automated staining system.

The Abcam Iron Stain kit was far superior to the reagents we typically prepare, with increased working solution stability, and a staining time that was reduced by approximately 30-60 minutes.


Developing assays involving many potentially interacting factors can be prohibitively complex for investigators, but recent advances in IHC have reduced the challenges encountered during multicolour immunoenzymatic procedure development. These innovations give researchers the ability to better investigate combined variables with a method that gives them the advantage of being able to reference materials indefinitely.

Multicolour IHC allows researchers to visually identify specific cell types/populations or determine cell derivation, and also identify specific processes happening within those cells, in well-preserved, whole tissue specimens of various states. The ability to utilise standard histological stains in conjunction with IHC staining gives researchers additional benefits in examining pathogenesis in tissues.

The assay developed here has allowed our researchers to investigate aspects of apoptosis in human macrophages of different tissues containing excess iron, in a manner which previously would only have been possible with multiple serial tissue sections stained separately for the IHC or IF markers, and the standard iron stain.

Visualisation of multiple markers in FFPE specimens with immunoenzymatic chromogens, as well as standard histologic stains, is a very powerful research tool.

The authors are: Shenna L Washington, Pamela Y Johnson, Mary D Beauchamp, Priya Handa Histology & Imaging Core Laboratory, Benaroya Research Institute, Seattle, WA & Abcam; Augustine Mzumara, product manager, Abcam





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