Lab Expectations

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. 

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.

Early exploration of unmapped biological signaling pathways were carried out using radiolabeled phospho-imaging. The development of phospho-specific antibodies to detect and quantify protein phosphorylation made life easier for researchers (less ³²P waste to deal with), but the interpretation of data from these experiments comes with its own set of caveats. 

Part four of a series on immunofluorescence techniques. Check out previous posts on Validation, Experimental Controls, and Fixation/Permabilization. 

After your samples have been prepared, it's time to incubate them with a well-validated antibody,  the workhorse of immunofluorescence. If you are a seasoned pro at IF experiments, you are probably used to checking the antibody datasheet (or web page) for the recommended dilution. But have you ever wondered where those recommendations come from?

Part Three of a four-part series on Immunofluorescence. Check out The Importance of Validation and Experimental Controls.

The performance of an antibody is a crucial determinant in getting reliable immunofluorescence (IF) results. Equally important is the preparation of the biological sample - cells or tissue used in your experiments - before any antibodies are introduced. The fixation and permeabilization of your samples are key steps that can determine your experiment’s failure or success. The ideal fixative preserves a “life-like” snapshot while quickly stopping the degradative process of autolysis by crosslinking and inhibiting endogenous enzymes. This post provides examples of how different antibodies perform at their best using different protocols.

 Part two of a four-part series on Immunofluorescence. Check out our posts on Validation and Fixation and Permeabilization. 

After months of hard work, your research has honed in on a hypothesis you can test with immunofluorescence (IF). You've chosen antibodies and performed pilot IF experiments (see The Importance of Validation), and the localization of the protein appears reasonable. But how can you be sure the IF data you've acquired represents real biological phenomena? We present two examples of experimental controls in this post.

Part one of a four-part series on Immunofluorescence. Check out Experimental Controls and Fixation and Permeabilzation.

After months of hard work, your research has zeroed in on a hypothesis you can test with immunofluorescence (IF). But now you have to make a choice. How do you decide which antibody to use to get reliable IF results? How do you know if the images are accurately reporting the target's localization? We explore some considerations in this post.

Antibodies targeted to histone modifications may bind non-specifically to similar, but off-target histone modifications. Conversely, their specific binding can be inhibited by steric hindrance from modifications on neighboring residues. Assays like ELISA, western blot, ChIP, and IF are commonly used to demonstrate antibody specificity and sensitivity, but they cannot clearly predict how an antibody will interact with nearby epitopes. As a result, an alternative approach is needed when trying to validate an antibody to a histone modification target. Continue reading to see how we do it.