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.
So you've set the timer to five minutes for the first of three TBST washes for your western blot membrane. Now what? Sure, you could check your email or social media for the 30th time before lunch. Or you could do something informative, like check out a CST Tech Tips video! This is a new short video series featuring the same scientists who develop and validate CST antibodies, here to offer insights and protocol tips.
I’m a Product Scientist at Cell Signaling Technology (CST). My typical work day no longer looks like my fellow scientists’ daily grind, and at times, I even find it difficult to describe in words what I do, but I’ll give it a shot!
Our previous blog post, “Painless Publication: How to Write a Journal Abstract,” walked you through the steps in writing the abstract for a journal article. Now we turn our focus to writing abstracts for conference proceedings. Although there are some similarities between these two types of abstracts, there are also some distinct considerations and approaches for conference abstracts.
Have you ever sat frozen at the keyboard, facing the due date for your abstract and asking yourself how you can condense all of this information into 200 words or less? If this scenario resonates with you, rest assured you are not alone. There is a learning curve for writing an effective abstract, and thankfully there are also some tips that will help you get started and finish strong.
You are all amped up to run a western blot to identify “your favorite protein.” The lysates have been run and proteins separated by SDS-PAGE. Now it’s time to transfer proteins from the gel to the membrane, and you’re sitting wondering….wet or semi-dry?? Or maybe you are better prepared than I was as a graduate student and you already know your next step, in which case you are aware of the pros and cons of wet and semi-dry transfer.
So your experiments and data are funneling you down an inescapable path. You need to show direct gene regulation by your protein of interest. You think to yourself, “Oh, ChIP...”
You’re gathering data from all your experiments and preparing to present to your advisor and thesis committee at your annual progress report. You have an interesting hypothesis, and you have a validated antibody that recognizes your target protein on a western blot (WB). The molecular weight of the band is correct, and the expression of the target protein changes just the way you predicted it would. Now, you know — and you’d bet the house on it — when that powerpoint slide comes up, someone on your committee is going to ask about loading controls.
The use of multiple antibodies in a single experiment can provide useful information to researchers. Co-staining with multiple antibodies and cellular dyes is a simple, low-content form of multiplex analysis. Techniques for performing multiplex analyses in cells and tissues are powerful research tools that are applicable to general cell biology studies as well as diagnostic purposes. These techniques allow researchers to detect multiple biomarkers to assess their samples. They also allow for easy colocalization studies to determine relationships between analytes. Here we describe two common techniques for fluorescent staining using multiple antibodies in the same assay.
A picture is worth a thousand words, or in the case of immunofluorescent imaging, a thousand proteins. The images used to illustrate a scientific experiment should convey as much information as the text itself. Here at CST, we pride ourselves in the quality of our antibodies and our rigorous validation process. When we approve our primary antibodies for IF, we like to showcase them using high quality images generated in-house. Beyond our recommended IF protocols (check it out here), here are some additional considerations to make when planning your IF staining.