Successful Immunofluorescence: The Importance of Validation


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

IF_Brochure_pt1_Blog_header_1600x200.jpgPart 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.

The primary antibody is a critical component of an IF experiment, and its performance directly affects data quality. Detection of a specific band on a western blot (WB) is not sufficient to guarantee a chosen antibody will perform in IF. WB analysis subjects proteins to harsh reducing/denaturing conditions that alter protein structure, so an epitope recognized by an antibody approved for WB may be buried and/or inaccessible for IF, where proteins remain in their native state.

Validate Specificity to Avoid Misleading IF Results

The α-Synuclein protein is highly expressed in the brain and its dysfunction plays a role in neurodegenerative diseases, such as Parkinson’s disease. In healthy tissue, α-Synuclein is expected to be localized to presynaptic terminals where it associates with synaptic vesicles. CST scientists compared α-Synuclein (D37A6) XP® Rabbit mAb #4179 side-by-side with another company’s α-Synuclein antibody, using each at the manufacturer’s recommended dilution. Both antibodies performed as expected in WB. In IF, punctate staining consistent with presynaptic localization of α-Synuclein was observed in midbrain sections for #4179, while the other company’s antibody showed a less punctate pattern, but critically mis-reported localization in nuclei or soma (arrows).

 IF_Brochure_pt1_validation_image_rpl_1200x600.jpg

A “clean” WB is not sufficient to ensure performance and reliability of an antibody in IF analysis. WB analysis of extracts from mouse and rat brain using α-Synuclein (D37A6) XP® Rabbit mAb #4179 or the other company’s antibody to α-Synuclein (left). Confocal IF analysis (right) of [mouse] lower midbrain and hippocampus sections using #4179 (upper row) or the other company’s antibody (lower row). Neuronal soma/ nuclei that are mis-labeled for α-Synuclein with the other company’s antibody are noted with white arrows.


This experiment illustrates the importance of application-specific validation. 

Check back in the coming weeks, we'll have posts discussing the design of controls for IF, considerations for fixation and permeabilization, and antibody incubation conditions. You can also follow the link below to download the Guide to Successful Immunofluorescence, a handy resource packed with tips and the 9-step Protocol for a Successful Immunofluorescence Experiment.

Download the Guide

Already have a copy of the guide? Check out these other IF posts:

5 Steps to Publication-Worthy IF Images
3 Questions to ask before starting your IF experiment
Fluorescent Staining Using Multiple Antibodies

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