Lab Expectations

Understanding the microenvironment of disease is critical. Targeted cancer therapies may not be effective depending on the combination of cell types in the tumor. Likewise, an immune cell invasion in pancreatic tissue could indicate the progression of type 1 diabetes. For years, this complex landscape could only be witnessed in single-cell snapshots by flow cytometry or limited tissue staining. Imaging Mass Cytometry™ (IMC™) now offers a panorama of this dynamic world.

For decades, immunohistochemistry (IHC) has been a powerful technique for the investigation and visualization of cellular components in their native histological context. IHC has served as an important tool in medicine – enabling the diagnosis of complex pathological conditions – and in basic research to advance the understating of key biological processes. 

Careful planning and the fine tuning of experimental protocols are key to ensuring clear, interpretable scientific results. This is especially true for immunohistochemistry (IHC) studies, where each step in the often multiday process – from tissue preparation to stain development – can significantly impact the final outcome and analysis. Often, the simultaneous examination of multiple antigens is required to address specific scientific questions, which further complicates IHC protocol development. A general understating of the steps necessary to optimize IHC for multiple targets is essential to achieve reliable results. So, what are these steps?

Immunohistochemistry, or IHC, remains the simplest method for detecting biomarker expression while maintaining spatial context within tissues. You know that getting reliable IHC staining results hinges on the specificity and performance of your antibody. These are high stakes experiments, and you want to be 100% confident your antibody will detect the target of interest.

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For generations of neuroscientists, using immunohistochemistry to study the brain in its anatomical context typically meant imaging a tiny slice at a time. Using the traditional method of taking micron thick sections, fixing, staining, imaging, and, finally, stitching all the slices together, is a super laborious task, especially for large tissues. But what if you could "look" into an intact mouse brain and identify specific cells and eliminate all of the slicing and stitching?

In recent years, immune checkpoint proteins in the tumor microenvironment have been under intense study. If you work in the immuno-oncology field, chances are you are either performing multiplex IHC (mIHC) or would like to. Ultimately, a multiplexed image like the one featured here provides a multi-layered depiction of a tumor, such that each antibody corresponds to a different fluorescent signal. If you want to detect more targets in your IHC, but aren’t sure how to design a panel of antibodies and fluorophores for mIHC, we’ll walk you through the process in this post.

If you’ve ever transitioned your IHC experiments from a manual protocol to an automated platform, you may have found the conversion process to be a drag. It isn’t an easy thing to do. For that reason, we’re happy to announce our IHC Leadership in Automation initiative. This rigorous validation initiative expands on our already thorough measures, allowing researchers to not only use CST products with our recommended manual IHC protocol, but also to bridge the assay to new platforms and techniques. Our foray into the world of automated IHC aims to reduce the amount of time researchers spend on assay transfer and protocol optimization.

The study of the tumor ecosystem and its cell-to-cell communications is essential to enable an understanding of tumor biology, to define new biomarkers to improve patient care, and ultimately to identify new therapeutic routes and targets.

This is part two of a two-part series on how to optimize your IHC protocols. Part one introduced the principles behind antigen retrival. Click here if you missed it... but, if you've got your tissue prepped and ready to go, we'll move on to the next steps in the staining protocol.

It’s Friday night and you could be out with your friends right now, but instead you’re tucked away in a dark little room filled with microscopes. Spending the evening in the lab seemed like a good choice at the time because you were certain this immunohistochemistry was going to reveal some small - but important - mystery of the universe to you. But now you’re sitting here, cursing the universe and everyone in it, because all you see when you stare down into the scope is some indistinct fuzziness. And did the controls work - meh - who’s to say? There’s no sugar coating it. It’s a fail.