BIOTECHNOLOGY 59
Te 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.
Te 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. Tis 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. Terefore, 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.
Te 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.
Te 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.
Conclusion 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. Tese 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. Te ability to utilise standard histological stains in conjunction with IHC staining gives researchers additional benefits in examining pathogenesis in tissues.
Te 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.
www.abcam.com/IHC
www.scientistlive.com
“Improved reagents simplify multicolour enzymatic IHC assay development for Formalin- Fixed Paraffin- Embedded tissues.”
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