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

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Materials for Chromogenic Detection on FFPE Tissue

ISH Reagents from ACD


IHC Reagents

RNAscope® 2.5 HD Reagent Kit-RED (Cat No. 322350) which includes:


Normal Serum Blocking reagent

HD Detection Kit (RED) (Cat# 322360)


Green HRP Chromogen

Wash Buffer (Cat No. 310091)


IHC Validated Primary Antibody

Target Retrieval Reagent (Cat No. 322000)


HRP- Polymer Conjugated Secondary Antibody

Protease and Peroxidase Blocking Reagent (Cat No. 322330)


Gill’s Hematoxylin I (50%)


VectaMount™ Mounting Medium (or equivalent)

ISH Staining with ACD’s 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 ACD.

Examples of Tissue and Target-Specific Pretreatment Condition Recommendations

Target Antigen Retrieval Protease Treatment
CD68 15min at 99°C in Oster® Steamer 15min at RT
FoxP3 15min at 99°C in Oster® Steamer 15min at RT
CD8 15min at 99°C in Oster® Steamer 15min at RT
Gata3 15min at 99°C in Oster® Steamer 15min at RT
Vimentin 15min at 99°C in Oster® Steamer 20min at RT
CD52 15min at 99°C in Oster® Steamer 20min at RT

*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.

      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.

  1. Wash slides twice for 2 minutes in wash buffer.
  2. Incubate tissue sections for 15 minutes in 200µL of Normal Serum Blocking Reagent. Tap solution off tissue sections- do not rinse slides.
  3. Using experimentally predetermined optimal conditions, incubate slides in 200µL of primary antibody solution.
  4. Wash slides twice for 2 minutes in wash buffer.
  5. Apply 200µL of HRP polymer-conjugated secondary antibody and incubate for 30 minutes.
  6. Wash slides three times for 2 minutes each in wash buffer.
  7. 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.
  8. Apply 200 µL of green HRP solution to slides and incubate for 10-15 minutes.
  9. 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.

  10. 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.
  11. Once dry, use a small amount of media to mount tissues.

Tonsil tissue probed for use in dual RNAscope ISH-IHC application displaying both RNA and protein expression of CD31/PECAM1.

Example of dual RNAscope ISH-IHC probed tissue. Formalin-fixed paraffin-embedded tissue sections of human metastatic tonsil were probed for CD31 mRNA (ACD RNAScope probe, catalog # 487381; Fast Red chromogen, ACD catalog # 322500). Adjacent tissue section was processed for immunohistochemistry using mouse monoclonal CD31 (Novus, catalog # NB600-562) at 1:25 dilution for 1 hour at room temperature followed by incubation with the anti-mouse IgG VisUCyte HRP Polymer Antibody (Novus, Catalog # VC001) and DAB chromogen (yellow-brown). Tissue was counterstained with hematoxylin (blue).