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SpeakEasy Science Blog

Tips Tricks 8

The Waiting Game: A Search for a Streamlined IHC Protocol

Picture this: a chemist walks into a lab and asks his colleagues how to transition some immunohistochemistry (IHC)-stained slides in a buffer solution to coverslip with a non-aqueous mounting media. From his perspective, it’s a simple problem, and it should have a similarly simple solution. Does it? Read this blog article to find out.

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Tips Tricks 10

Dial up the Contrast with Counterstaining

When scientists detect targets of interest with immunohistochemistry (IHC), antibody binding and substrate detection alone often produce a perplexing picture where the target lacks context. Luckily there’s a solution. Counterstaining IHC samples after enzymatic detection spatially orients the target within its surroundings, which can help guide researchers as they analyze their results.

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Tips Tricks 11 1

A Simple Guide to Immunohistochemistry Substrate Selection

When it comes to selecting the substrate for your IHC application, you have variety and flexibility, and our focus is to help you make the right decision. This blog discusses different factors to consider—enzyme, sensitivity, color, method of visualization, and stability—when selecting the appropriate substrate (and, consequently, color) for your experiment.

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Tips Tricks 9

An Introduction to Bioconjugation: Answering Your Questions

Bioconjugation offers a way to chemically link two molecules to form a single hybrid, where at least one of the molecules in the partnership is a biomolecule. The resulting product retains the activity of each component, yet also gains novel functionality that is not possible with either molecule alone. In a recent webinar, Dr. Craig Pow, Director of Technical Services at Vector Laboratories provided an introduction to bioconjugation and its applications, as well as key factors to success.

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Tips Tricks 13

Reduce TissueBetter Immunofluorescence at the Speed of Light

When everything goes right, looking through the microscope at your immunofluorescence-stained tissue is no less beautiful than peering through a telescope at the night sky. Unfortunately, experimental issues can obscure that pristine view of your microscopic “galaxy” so much that that you can’t tell the difference between Halley’s Comet and the Big Dipper! Never fear, we’re here to help.

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Tips Tricks 14

The Curious Case of the Staining that Went Wrong

There’s nothing worse than toiling away all week to repeat an experiment only to have your staining turn up blank. You’re left scratching your head trying to figure out why you’ve got nothing. Recently, Vector Laboratories’ very own Sherlock Holmes, Dr. Craig Pow, joined The Scientist University to discuss Improving IHC and IF Staining Results.

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Tips Tricks 10

Improving IHC & IF Staining Results

Immunohistochemistry Immunofluorescence Immunohistochemistry (IHC) and immunofluorescence (IF) are widely used across the life sciences—in cancer research, immunology, neuroscience, cell biology, and histopathology—to help elucidate protein expression in cell and tissue specimens. And while these are well established and widely adopted techniques, when working with biological specimens, you can encounter common problems, such as background interference, lack of specificity, weak signal, or poor reproducibility. In a recent webinar with The Scientist, Dr. Craig Pow, presented helpful tips and tricks for improving IHC and IF staining results. You can watch the full on-demand webinar or keep reading for some tips and tricks from the Q&A session. How do the sensitivities of IHC and IF compare? While the workflows for IHC and IF do have several similarities, it is can be a little bit like comparing apples and oranges. It is exceedingly difficult to draw a straight-line comparison between the two different applications. Immunohistochemistry can be executed with a one- or two-step methodology followed by enzyme substrate detection with signal amplification, all while providing greater sensitivity with the two-step method. Conventional immunofluorescence on the other hand, provides exceptionally fine, exquisite detail, depending on what you need to examine and hope to resolve. /*! elementor – v3.18.0 – 08-12-2023 */ .elementor-widget-image{text-align:center}.elementor-widget-image a{display:inline-block}.elementor-widget-image a img[src$=”.svg”]{width:48px}.elementor-widget-image img{vertical-align:middle;display:inline-block} For example, when studying subcellular localization, with multiplexing and immunofluorescence, you can easily colocalize expression of antigens in the same cell compartment of the same cell. You also can choose to implement amplified immunofluorescence approaches to increase sensitivity, for instance by opting to use VectaFluor™ Excel antibodies that can increase sensitivity by as much as three- to four-fold over fluorophore-conjugated secondary antibodies. When it comes to detection, why should someone choose an alkaline phosphatase IHC detection method over one with horseradish peroxidase? The use of peroxidase-based methodology, alongside a substrate such as 3,3′-diaminobenzidine (DAB), is probably the most used approach. It deposits a nice discrete colored precipitate for single protein detection. However, if you are working with tissue that contains endogenous peroxidase activity, such as highly vascularized material, you may need to add a quenching step. Quenching of the endogenous peroxidase can be managed by incubation with hydrogen peroxide solutions (3% H2O2 in water, 0.3% H2O2 in methanol) or commercial convenience products, such as BLOXALL® Endogenous Blocking Solution, Peroxidase and Alkaline Phosphatase. While quenching can be effective, it does add more steps to the staining process. For some tissues, switching to an alkaline phosphatase approach can circumvent the issues of persistent peroxidase activity. As an added benefit, the diffuse and translucent color of alkaline phosphatase substrates can better provide a clear view of the underlying tissue morphology. Alkaline phosphatase (AP) detection also provides a choice of several colors. For example, if you’re working with tissue with inherent pigmentation or melanoma, additional substrate color options for alkaline phosphatase detection enable broader spectrum staining in magenta, blue, indigo, and black. You can leverage these colorful options with double or triple staining. Explore protocols for chromogenic multiplexing in our IHC Multiplexing Guide. How can I manage autofluorescence in my IF tissue preparation? Autofluorescence or endogenous fluorescence may be inherent to your tissue or preparation method. It may arise from different cell types or multiple factors. For example, collagen, elastin, and red blood cells are particularly notorious for autofluorescence. Aldehyde-based fixatives, such as formalin, paraformaldehyde, or glutaraldehyde can induce autofluorescence. TrueVIEW® Autofluorescence reagents help diminish unwanted autofluorescence and dramatically improve signal-to-noise ratio. TrueVIEW reagent is typically used after the secondary antibody incubation and subsequent washes. The reagent is applied for about two to five minutes, and then the sections are washed with PBS prior to mounting with anti-fade media. Is it better to wash for a) fewer times for longer periods of time or b) more frequently for shorter periods of time? While there’s no definitive answer for this, you will need to choose whatever is effective to help reduce toning, background, and remove the unbound material from your tissue preparation. At Vector Laboratories, we usually run two to three washes (2-5 minutes each) in between incubations. If you use thicker tissue sections, longer washes are required. Three to five washes (10–20 minutes each) are not unheard of for thicker sections, embryos, Xenopus, or zebrafish. Your best approach will start with these recommendations and optimize the steps to see what works best for you. What other negative controls would be appropriate, beyond substituting nonimmune IgG? Probably the best negative control for most would be to apply an irrelevant antibody, meaning something that will not bind to anything in your tissue preparation. Let’s say that you work with NeuN or GFAP on CNS or brain tissue. You know there won’t be any stratified keratinized epithelium in your tissue prep. In this scenario, you could select a primary antibody against stratified keratinized epithelium, raised in the same species as the isotype, at the same concentration, and then use typical detection reagents after that. This negative control would show that the primary antibody isotype from that species is not being bound inadvertently by some tissue or contributing to the staining. Other negative control options may be used, such as if you’re researching a knockout model and you can test your antibody on that tissue. Or pre-immune serum, which is serum extracted from the animal prior to antibody immunization, could be applied instead of the primary antibody as a validation control to check for non-specific binding. Another control would be to incubate the primary antibody with the immunogen used to generate the antibody, but this approach may not be practical for those who use commercial antibodies. Watch our on-demand Technique Talk webinar for more tips on identifying and eliminating sources of non-specific staining, as well as the appropriate controls to use. And be sure to check out our IHC Resource Guide and IF Resource Guide to help you navigate the workflows and select reagents. Got more questions? Check out our FAQs or connect with an expert to learn more.

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