ISH-IHC Protocol for Chromogenic Detection on Formalin Fixed Paraffin Embedded (FFPE) Tissue

Materials for Chromogenic Detection on FFPE Tissue

ISH Staining with RNAscope Assay

See RNAscope 2.5 HD Reagent Kit-RED Assay User Manual. The pretreatment conditions for FFPE tissues outlined below for Target Retrieval step are only recommendations. It may be necessary to optimize protease digestion for the IHC target of choice. Always follow user manual protocols that correspond to their appropriate kits purchased from Bio-Techne.

Examples of Tissue and Target-Specific Pretreatment Condition Recommendations

*These recommended incubation times are for FFPE tonsil tissue. For less dense tissue (i.e. breast, normal lung, colon), decrease protease incubation time. For denser tissue (i.e. liver, muscle, or highly expressed protein targets (Vimentin)) longer protease incubation time is required.

IHC Staining

IHC staining should be performed immediately following ISH chromogenic detection.

1. • Note: PBS buffer decolorizes green chromogen. When using green chromogen, use only wash buffer provided by ACD for all washing steps. All steps are done at room temperature unless specified differently.


2. Wash slides twice for 2 minutes in wash buffer.
3. Incubate tissue sections for 15 minutes in 200 µL of Normal Serum Blocking Reagent. Tap solution off tissue sections - do not rinse slides.
4. Using experimentally predetermined optimal conditions, incubate slides in 200 µL of primary antibody solution.
5. Wash slides twice for 2 minutes in wash buffer.
6. Apply 200 µL of HRP polymer-conjugated secondary antibody and incubate for 30 minutes.
7. Wash slides three times for 2 minutes each in wash buffer.
8. While your slides are washing, make a 1:50 working solution of a chromogen by mixing Green B (1 µL) with Green A components (49 µL). Make enough for 200 µL per slide.
9. Apply 200 µL of green HRP solution to slides and incubate for 10-15 minutes.
10. Quickly tap green HRP solution off slides and briefly rinse with distilled water.
    • Note: Green substrate can lose its color by extended exposure to water.
11. Counterstain with 50% Hematoxylin I for 30 seconds. Gently rinse slides briefly with distilled water and place on bench top to dry or use oven to speed up drying procedure.
12. Once dry, use a small amount of media to mount tissues.