multiplexing | Visikol

The Importance of Dehydration and Rehydration of Fixed Tissue for Immunolabeling

Carol Tomaszewski — Wed, 10 Aug 2022 13:06:56 +0000

In order to optimize the quality of microscopic images obtained from Formalin Fixed Paraffin Embedded (FFPE) samples, the starting condition of the fixed tissue must mimic the organic tissue as closely as possible. In order to achieve this, the tissue must first be fixed as soon as possible after removal from the specimen. Fixing the tissue properly preserves it from decay and halts all ongoing biochemical reactions.

A properly de- and rehydrated kidney sample labeled for α-Tubulin (green) and nuclei (blue)

After fixation, the tissue must be dehydrated to allow for complete penetration of the tissue by the paraffin wax. Since wax is not soluble in water, but is soluble in xylene, the water in the tissue must be replaced with xylene. Unfortunately, xylene is also not soluble in water but is soluble in alcohol. The sample must be gently dehydrated by immersing the samples in a graded series of alcohol solutions, ending with pure 100% reagent or isopropyl alcohol and then xylene. If one were to simply immerse the sample in 100% alcohol, degradation of the sample and proteins could occur. Improper dehydration can result in incomplete saturation of the sample with paraffin wax and, consequently, softness of the tissue.

Softness of the tissue can lead to uneven slices, tears, or ripples in the tissue and suboptimal, or even unusable, images after labeling or staining. After a usable slice of tissue is obtained, ironically, the tissue must be rehydrated. Most staining and labeling solutions and protocols are aqueous and thus, the wax must be removed again from the sample mounted on the slide. This process is essentially the opposite of the dehydration process and, when done properly, allows for complete infiltration of the sample with the chosen stain or immunolabel. If this process is done improperly, this can result in incomplete infiltration and as a result a false negative result.

Visikol and its cutting-edge Multiplex technology utilizes immunofluorescence techniques and standard immunohistochemistry methods which offer a new paradigm in research, opening a whole new door to understanding the tissue microenvironment, disease progression, and treatment. If you’d like to learn more, visit our Multiplex Technology Page, our Overview Page, or our recent blog post about the Clinical Applications of Multiplex Imaging and how it can be utilized as a Clinical Tool.

Please reach out to a remember of our team if you’re interested in learning more about Multiplexing technology.

The post The Importance of Dehydration and Rehydration of Fixed Tissue for Immunolabeling first appeared on Visikol.

Autofluorescence Quenching

Carol Tomaszewski — Wed, 08 Sep 2021 12:21:56 +0000

Autofluorescence has been a recurrent problem in immunofluorescent staining. It impedes on distinguishing specific signaling vs. non-specific (background) in tissues that contain natural fluorescence from endogenous fluorophores. Collagen, elastin, flavins, red blood cells and lipofuscins are known to have natural endogenous fluorescence. A tissue that contains any of the above components in images are prone to have miniature speckles that have a strong fluorescent excitation, anywhere between 360nm to 647nm. Autofluorescence can also hinder data analysis by making it difficult to co-localize proteins due to specific signaling being masked. Fortunately, several autofluorescence quenchers have been developed to bypass unwanted non-specific signaling from endogenous fluorescence.

What Is Autofluorescence Quenching?

Autofluorescence quenching is the process in which the intensity of emitted light from fluorescent molecules is decreased. Three common quencher kits discussed today will be TrueVIEW, TrueBlack and Sudan Black b.

TrueVIEW diminishes unwanted autofluorescence from non-lipofuscin sources (collagen, elastin and red blood cells) in tissue sections that have an aldehyde-based fixative. It can be stored at room temperature and/or in the fridge for 48 hours and should be discarded after. TrueVIEW will stain tissue sections blue, but it should not interfere with immunofluorescence labeling. It is most effective in kidney and spleen tissue.
TrueBlack is primarily used to quench lipofuscin autofluorescence. It can improve background from other sources, such as collagen, elastin, and red blood cells. It can be stored at room temperature in a dark place for 12 months. It is most effective in human brain and retina tissue.
Sudan Black b, also known as Solvent Black 3, acts as a dark mask to diminish lipofuscin autofluorescence and other sources without covering immunofluorescence labeling. It comes as a powder so it can be stored at room temperature long-term. It is known to be effective in multiple tissue types, such as pancreas, kidney, brain, mandibles, etc.

All three common quenchers must avoid any washes that contain detergent, as it will wash the dye away. Autofluorescent quenchers have enhanced immunofluorescent staining, allowing Visikol to visualize specific proteins and structure.

What Does Visikol Use for Autofluorescent Quenching?

At Visikol, we use Sudan Black b as our autofluorescent quencher. We prepare it in 0.3% of the Sudan Black b powder in 70% ethanol overnight on a shaker in the dark. The next morning, it is filtered and ready for use. Incubation is done after immunolabeling for about 10-15 minutes depending on the tissue type. Visikol has chosen to use Sudan Black b due to documented validation of multiple tissue types and it’s compatibility with our multiplexing and striping protocols. Visikol works closely with clients to help them meet their drug discovery goals. Reach out to us today to chat with our team if you have any questions or want to know more about our services.

Without Sudan Black b

With Sudan Black b

The post Autofluorescence Quenching first appeared on Visikol.

Leica Biosystems and Visikol to Host Multiplex Webinar

Griffin Ferrara — Tue, 09 Feb 2021 17:21:42 +0000

Visikol CEO Dr. Michael Johnson will be hosting a webinar with Leica Biosystems discussing multiplex imaging and the Leica VERSA 8.

Through this presentation, Visikol will provide a background on various multiplex tissue imaging approaches and will describe how they can be used to answer complex research questions such as surveying the immune cell population within a tumor microenvironment or evaluating therapeutics for infectious diseases. Specifically, Visikol will discuss how its proprietary multiplex approach has been leveraged through a partnership with Hackensack Meridian Health (HMH) to evaluate tuberculosis treatments. This work is funded by the Bill & Melinda Gates Foundation using Visikol’s multiplex approach combined with several other imaging modalities to evaluate treatments using a multi-modal imaging approach.

There are three main learning objectives of the presentation:

Develop an understanding of multiplex slide labeling and imaging techniques.
Demonstrate a working knowledge of the multiple types of multiplex tools available for whole slide imaging.
Identify the unique types of research questions that can be addressed through multiplex imaging

The webinar will be on 3/25 at 8am Pacific, 11am Eastern, 5pm Central Europe, 4pm UK with a live Q&A afterwards.

Watch the Recorded Webinar

The post Leica Biosystems and Visikol to Host Multiplex Webinar first appeared on Visikol.