Discover essential techniques for successful multiplexing immunohistochemistry (IHC) in this informative webinar presented by neuroscientist and microscopist Darpan Saraswat. Learn how to optimize sample preparation, prevent autofluorescence, and minimize unspecific antibody labeling in complex samples like tumor microenvironments. Explore strategies for selecting appropriate markers, fluorophore dyes, and labeling techniques to expand your IHC experiments. Gain insights into avoiding fluorescent bleed-through and spectral overlap, and understand the importance of controls in multiplexed IHC. Master the art of planning and executing larger Spatial Imaging panels while considering factors such as fluorophore photobleaching and photostability. Enhance your skills in advanced microscopy methods and high-resolution imaging techniques to elevate your research in cellular analysis.
Overview
Syllabus
Intro
Multiplexed immunohistochemistry (mIHC)
Optimization of sample preparation
Blocking autofluorescence
Tips for blocking background
How to plan a successful multiplexed IHC experiment
How to choose an appropriate primary antibody?
The Thermo Fisher Scientific antibody search tool
Primary antibody protocol optimization
How do you choose an appropriate secondary antibody?
Secondary antibody protocol optimization
Types of labeling strategies
How to decide appropriate labeling strategy?
Direct conjugates of primary antibody
Standard secondary antibody labeling
Avidin-biotin amplification
Enzyme amplification
Points to remember for labeling strategy
Multiplex fluorescence: The main colors and beyond...
How to choose the optimal fluorophore and filter?
Spectral overlap and multiplexing
Fluorophore photobleaching and photostability
Controls and multiplexing
Some important rules to remember for multiplexed IHC
Taught by
Labroots
