Faqs

Under-fixation of tissue specimens will result in protease over-digestion, which leads to loss of RNA and poor tissue morphology. Over-fixed tissue specimen will result in protease under-digestion, which leads to poor probe accessibility and low signal and signal/background ratio while maintaining excellent tissue morphology.

ACD recommends tissue fixation in accordance with standard clinical research guidelines. FFPE samples should be fixed in FRESH 10% NBF (neutral buffered formalin) for 16 – 32 hrs at RT. NOTE: Do not fix at 4°C. Do not fix for < 16 hrs or > 32 hrs. Delayed fixation can degrade RNA and produce lower signal or no signal. Shorter time or lower temperature will result in under-fixation. For optimal results, ACD highly recommends using the 10% NBF tissue fixation methodology.

ACD always recommends starting by establishing a working IHC protocol and then using the same established secondary for ICW. You may use any secondary you have been using in the lab for your IHC workflow.  Please ensure that any secondary that you choose uses the appropriate detection chemistry (e.g. HRP-conjugated secondary for ICW, fluorescent-conjugated or HRP-conjugated secondary for fluorescent detection).

With the increased compatibility of the new integrated co-detection workflow across a wide variety of antibodies, there are no specific antibody clone recommendations. ACD encourages you to use your preferred clones for RNA-protein co-detection. For best results, titrate your preferred primary antibody concentrate in ACD’s Co-Detection Antibody Diluent in the context of the integrated co-detection workflow.

Co-Detection Blocker prevents cross-detection of RNAscope signal by the IHC detection steps.

The ACD Antibody diluent has been formulated to ensure maximal retention of RNA sample quality.

Our sequential dual ISH/IHC workflow allows for IHC staining following RNAscope. Protease pretreatment is necessary for RNAscope but can impact your target protein of interest. Some epitopes may be better conserved than others after protease treatment and some optimization of protease may be necessary to find an optimal condition that allows for both RNAscope and IHC detection. The new integrated co-detection workflow cross-links the primary antibody prior to the protease step, allowing conservation of the antigen/antibody for detection.

RNA/protein co-detection is compatible with the RNAscope 2.5 HD RED, RNAscope Multiplex Fluorescent V2, BaseScope v2, and miRNAscope HD (RED) assays.

Please contact Indica Labs directly at info@indicalab.com for HALO related questions.

You can analyze your image either semi-quantitatively or quantitatively. Please refer to the scoring guideline for semi-quantitative analysis or use image analysis software to analyze quantitatively. ACD has technical notes providing guidance on how you can use ImageJ/Cell Profiler/QuPath to quantify gene expression from your RNAscope images. In-house ACD uses HALO software from Indica Lab for quantitative analysis.