IF Video: 3 Questions You Should Ask

Before you start your next immunofluorescence (IF) experiment, watch this short video to learn 3 things you need to know to get reliable results and avoid headaches: 



[Narrator] Before starting an immunofluorescence experiment, you should ask yourself the following three questions:
  1. Is your antibody specific?
  2. Is your antibody supported by an optimized IF protocol?
  3. Is your antibody performing consistently?

Let's take them each in turn.

First, is your antibody specific? Wild-type mouse embryonic fibroblasts express two isoforms of Glycogen Synthase Kinase 3: GSK-3 Alpha and GSK-3 Beta. The Cell Signaling Technology antibody for GSK-3 Alpha detects endogenous levels of GSK-3 Alpha in wild-type cells, as well as in GSK-3 Beta knockout cells, as expected. This antibody exhibits no cross-reactivity to the Beta isoform of GSK-3 expressed in GSK-3 Alpha knockout cells, thus confirming its isoform specificity.

Is your antibody supported by an optimized IF protocol? Side-by-side comparison of HeLa cells, fixed using different fixation protocols, shows that Keratin 8/18 mouse monoclonal antibody works best with methanol fixation. In contrast, AIF rabbit monoclonal antibody works best with formaldehyde fixation. Save yourself from spending precious time in reagents on efforts to optimize a protocol that works.

At Cell Signaling Technology, we have determined the optimal fixation, permeabilization, and antibody dilution conditions for you. And finally, is your antibody performing consistently? Ensure reagents are reliable for the life of your project. We test every new antibody lot to ensure its performance is equivalent to that of previous lots. Stringent testing ensures lot-to-lot consistency.

How did your antibody do? If you answered yes to all three questions, then you should feel pretty secure that your antibody will support your experiments, and help you move your research forward. All Cell Signaling Technology, antibodies approved for use in IF have undergone a rigorous validation process, that includes verification of the correct sub-cellular localization and target-appropriate cell or tissue model systems.

Whichever antibody you choose, the Cell Signaling Technology technical team is always here to help you set up or troubleshoot your experiments. And please, click here if you'd like to learn more about how we validate our antibodies for IF, as well as other applications.

Cell Signaling Technology.

For Research Use Only. Not For Diagnostic or Therapeutic Purposes. © 2018 Cell Signaling Technology, Inc. CST and Cell Signaling Technology are Trademarks of Cell Signaling Technology, Inc.