The breast cancer TMA (US Biomax Inc.), including invasive ductal carcinoma cases (grade 2, n = 28; grade 3, n = 25) and medullary carcinoma cases (n = 9), was used for multiplex IHC staining of TRIM21, CD73, CD3, CD8, Ki67, and PanCK by using the Opal multiple color IHC kit (Akoya Biosciences) as described previously (53 (link)). Briefly, the deparaffinized and rehydrolyzed TMA slide was implemented antigen retrieval in AR9 retrieval buffer (Akoya Biosciences), followed by 6 cycles of staining procedures including blocking and binding of primary antibodies and second HRP-linked antibodies and visualized with the corresponding Opal fluorophores. Each staining cycle was finished up with heating in AR6 retrieval buffer (Akoya Biosciences) to release the bounded primary and second antibodies but did not disturb the resident fluorophores. After six-round staining procedures, the slide was counterstained with DAPI. The single-marker staining with individual opal fluorophore was used as the reference for the “spectral unmixing process.” The antibodies and corresponding fluorophores are listed in table S1.
Ar6 retrieval buffer
Manufactured by Akoya Biosciences
The AR6 retrieval buffer is a solution designed for use in immunohistochemistry (IHC) and other biological applications. Its core function is to facilitate the retrieval of target antigens from formalin-fixed, paraffin-embedded (FFPE) tissue samples, which is a crucial step in the sample preparation process for IHC analysis.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using ar6 retrieval buffer
1
Multiplex IHC Profiling of Breast Cancer
2
Multiplex Immunohistochemistry of Skin Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The fixed tissues were dehydrated for embedding in paraffin. Five-micrometer tissue sections were subjected to either hematoxylin/eosin staining or immunohistochemical study using an Opal 4-Color Multiplex IHC kit, according to the manufacturer’s instructions (Akoya Bioscience). Briefly, skin sections were deparaffinized, rehydrated, and refixed with 10% neutral buffered formalin prior to antigen retrieval by heating for 15 min in AR6 retrieval buffer (Akoya Biosciences). Next, the tissue sections were subjected to sequential staining procedures including blocking, binding of the primary antibody and HRP-labeled secondary antibody where HRP-labeled secondary antibody catalyzes activation of tyramide-fluorophore to form covalent bonds with the tyrosine residues present in close proximity of the protein of interest. The tissues were heated to remove the primary and secondary antibodies leaving the covalently bound fluorophores on the tissue, and another primary antibody was used for the next step of staining.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!