Immunofluorescence is a technically simpler method of visualising multiple antigenic markers.
Tese 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. Tese qualities of multicolour IHC are of significant value to researchers, especially in the early phases of study.
Tere 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.
Tese products reduce the challenges of involved substrate preparation, and significantly reduce the length of staining procedures. Te present work discusses how improved reagents simplify multicolour enzymatic
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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. Tis 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).
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