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  • Fluorescent imaging training part 3: protocols


    Welcome to our training series on fluorescent imaging. We’ll show you imaging basics and essential steps before moving on to optimization, troubleshooting, and more advanced techniques.

    In part 3 of the training series, we introduce you to some of the most important protocols. Our fluorescent immunocytochemistry/immunofluorescence (ICC/IF) and immunohistochemistry (IHC) protocols cover all aspects of your experiment, from sample preparation to immunostaining and mounting. Whether you want a protocol to follow in the lab during your experiment, or you want to understand in more detail what happens at each step, you can find the information below.

    Overview

    3.1 Fluorescent staining - quick videos
    3.2 IF and IHC protocols
    3.3 The IHC guide

    3.1 Fluorescent staining – quick videos

    Let's jump straight in with a quick overview of the key steps involved in fluorescent staining as well as considerations when investigating more than one antigen.


    ICC overview


    Staining more than one protein


    3.2 Step-by-step IF and IHC protocols

    This protocol provides you with the steps to follow for cell and tissue preparation, fixation, permeabilization, and fluorescent immunostaining. For the best results, we have also provided optimized protocols, and include recommended dilutions on the datasheets of all our antibodies. You can get a text version of this protocol here.


    3.3 A detailed look at IHC

    Fluorescent staining of tissue samples can provide higher resolution images and offers more potential to multiplex compared with chromogenic immunodetection. This comprehensive guide provides you with detailed information of each step in the immunofluorescent staining of tissue samples.


    Summary

    And that is the end of Part 3. You should now be fully versed in

    – ICC fundamentals
    – IF and IHC protocols
    – The finer points of performing IHC

    In Part 4, we show you how to deal with common problems encountered during ICC and IHC, like poor signal and high background, and how to carry out staining when using antibodies from the same species.