A Comprehensive Kit Designed to Facilitate the Simultaneous Execution of the BaseScope Red Assay Alongside Immunostaining Procedures

March 25, 2025

BlueskyReddit
State University of New York at Buffalo
Physiology and Biophysics
Postdoctoral Researcher

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Company:

Advanced Cell Diagnostics, Inc. (ACD)

Product Name:

RNA-Protein Co-Detection Ancillary Kit

Catalog Number:

323180

We regularly use this kit for the BaseScope Red Assay in combination with immunostaining as part of an integrated co-detection workflow. This approach enables us to simultaneously assess mRNA transcripts of interest, specifically Oprm1 (the gene encoding mu opioid receptors), and the protein expression of interest, which is the astrocytic marker GFAP, in fixed frozen mouse brain tissue.

Experimental Design and Results Summary

Application

RNA in situ hybridization, Immunostaining

Starting Material

15 μm fixed-frozen mouse brain sections

Tips

During the Target Retrieval step, the slides should be placed horizontally instead of vertically so that any active bubbling due to boiling will not interfere with the samples.

Results Summary

This assay using the RNA-Protein Co-Detection Ancillary Kit allows us to assess the expression of mu opioid receptors (mRNA) in astrocytes in different mouse brain regions of interest. This enables us to conduct quantitative analysis. The protocol requires calibration; for example, depending on tissue type, target retrieval boiling time needs to be adjusted. Image - two different samples (mouse nucleus accumbens); Scale bar - 20 µm. Images were obtained on an inverted confocal microscope Leica-Sp8 using LASXsoftware. The BaseScope Fast Red signal is fluorescent in the red channel (red punctate dots), and the GFAP (astrocytic marker) is in the green channel. Sections were counterstained with DAPI fluorescent dye (blue) to identify the brain subregions.

DOI or PMID #

N/A

Image Gallery

Summary

The Good

Once calibrated (the target retrieval step), the kit produces consistent good results. The packaging of the reagents is very convenient, which makes the whole process very easy and straightforward.

The Bad

The kit contains three reagents: Co-Detection Blocker, Co-Detection Antibody Diluent, and Co-Detection Target Retrieval. It would be better if the reagents were sold separately rather than as part of the kit. While there is a lot of Co-Detection Antibody Diluent (120 mL), the volume of Co-Detection Blocker is only 4.5 mL. Target Retrieval comes in four bottles (one bottle per assay). Therefore, you can run out of Co-Detection Blocker and Target Retrieval while still having a lot of Co-Detection Antibody Diluent. Selling the reagents separately might be more cost-efficient for the lab.

The Bottom Line

This kit ensures the success of the integrated (BaseScope + Immunostaining) workflow, allowing the detection of both mRNA transcripts and proteins of interest. It is very easy to use, and I highly recommend it. All reagents from the kit work as expected and produce robust results throughout the experiments.

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