Have you ever wondered: Am I setting up my loading controls correctly?


Posted by Ken B on Nov 1, 2017 3:00:00 AM

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.

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Topics: Protocols, Antibody Performance, Western Blot, Companion Reagents, techniques

Fluorescent Staining Using Multiple Antibodies


Posted by Tara W on Sep 13, 2017 3:00:00 AM


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.

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Topics: Protocols, Antibody Performance, Primary Antibodies, IF-IC, Companion Reagents, techniques

5 Steps to Publication-Worthy IF Images


Posted by Tara W on Sep 6, 2017 3:30:00 PM

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. 

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Topics: Protocols, Antibody Performance, Antibody Validation, IF-IC, techniques

Switching from Manual to Automated IHC? We've got your back


Posted by Chris G on Aug 30, 2017 3:00:00 AM

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.

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Topics: Protocols, IHC, Antibody Performance, Antibody Validation, techniques, Automated IHC

Intracellular Flow Cytometry in Action


Posted by Liana G on Aug 9, 2017 9:45:00 AM


Traditionally, flow cytometry has been used to identify distinct cell types within a heterogeneous pool of cells, based on extracellular or surface marker expression, an application commonly known as immuno-phenotyping. However, this technology is also readily amenable to intracellular target detection and can be successfully applied to the study of complex signaling events.

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Topics: Protocols, Flow, Cell Biology, techniques

4 Steps to Better Immunohistochemistry: Steps 2, 3, and 4


Posted by Carolyn P on May 31, 2017 3:00:00 AM

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.

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Topics: Protocols, IHC, techniques

4 Steps to Better Immunohistochemistry: Step 1 - Antigen Retrieval


Posted by Carolyn P on May 24, 2017 3:00:00 AM

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 the 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.

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Topics: Protocols, IHC, Companion Reagents, techniques

Successful Immunofluorescence: Antibody Dilution and Incubation Conditions


Posted by Ken B on May 10, 2017 3:01:00 AM

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?
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Topics: Protocols, Antibody Performance, Primary Antibodies, IF-IC, techniques

Successful Immunofluorescence: Fixation and Permeabilization


Posted by Ken B on Mar 15, 2017 3:00:00 AM

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.

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Topics: Protocols, Antibody Performance, IF-IC, techniques, Fixation

Successful Immunofluorescence: Experimental Controls


Posted by Ken B on Mar 8, 2017 3:00:00 AM

 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.

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Topics: Protocols, Antibody Performance, Primary Antibodies, Antibody Validation, IF-IC, Post Translational Modification, Reproducibility