Proteins exhibit astonishing diversity, enabling them to carry out myriad functions within living organisms. Within the human body are tens of thousands of various protein types, each with its unique function. Due to the overall diversity of proteins, scientists initially thought there must be at least 100,000 genes to encode all human proteins. However, researchers broke down our DNA when the Human Genome Project began and found a much humbler number—approximately 20,000 genes. This breakthrough challenged the notion that a higher gene count equals a higher organism complexity, highlighting the enigmatic interplay of genes and the intricate processes that govern protein diversity. But this left researchers with a lingering question: if there are so few genes, how are there many diverse proteins?

The Genetic Script: Transcription and Translation

At the heart of protein diversity is the process of translation, where the information encoded in the DNA is transcribed into messenger RNA (mRNA). This mRNA serves as a template for the synthesis of proteins. The journey begins with mRNA transcription from the DNA template using RNA polymerase. A process called splicing comes into play to generate diverse proteins from the same RNA template. The introns are excised during splicing, and the exons are joined together. This allows for different combinations of exons to be included or excluded, producing distinct mRNA molecules. The resulting mRNA variants, known as isoforms, provide the recipe for synthesizing diverse polypeptides. This mRNA transcript acts as the recipe for protein production in the process known as translation. During translation the mRNA is read by ribosomes and the corresponding amino acids are chained together to form proteins.

Post-Translational Modifications: Glycosylation, Phosphorylation, and Polyprotein Cleavage

The journey from mRNA to functional proteins continues after translation. Post-translational modifications add another layer of complexity, contributing to the incredible diversity of proteins. One common modification is glycosylation, where sugar molecules are added to proteins. A single protein can be glycosylated in many ways; the type of sugar added and where glycosylation occurs can significantly impact a protein’s role and function. This process not only influences the structure and stability of proteins but also plays a crucial role in protein transport, as specific glycosylated tags are needed for proteins to move between the endoplasmic reticulum and the Golgi apparatus of the cell. Another common post-translational modification is phosphorylation, which can activate or deactivate enzymes.

Additionally, some proteins may be derived from larger pro-protein molecules, allowing a variety of small proteins to be made from a much larger precursor molecule. These large molecules, called polyproteins, undergo cleavage to yield smaller, biologically active proteins, including hormones. An example is the prohormone pro-opiomelanocortin, which is cleaved to produce corticotropin and beta-lipotropin, each with its unique function. These post-translational modifications play a critical role in protein structure and function diversity. Proteins can be visualized in cells and tissues using immunohistochemistry (IHC) and immunofluorescence (IF) detection methods.

Harmony of Techniques: Immunohistochemistry (IHC), Immunofluorescence (IF), and RNA-FISH

Due to the diversity of proteins, there are various ways for scientists to visualize and analyze the distribution of proteins within tissues and cells. IHC and IF commonly utilize molecular methods to detect proteins within cells or tissues. The ability to recognize and differentiate between various protein isoforms and modified forms enhances our understanding of cellular processes and aids in identifying disease markers. These techniques allow scientists to visualize what proteins are present in normal tissues to compare protein expression patterns to those of diseased tissues. Adding another note to this symphony is RNA fluorescence in situ hybridization (RNA-FISH). This technique allows researchers to visualize and localize specific RNA molecules within cells, providing a dynamic perspective on gene expression. By combining IHC, IF, and RNA-FISH, scientists can create a comprehensive picture that unveils the intricate relationships between RNA and protein dynamics, enriching our understanding of cellular function and dysfunction.

The Crescendo: Molecular Biology and Medicine Knowledge

The journey from a single RNA template to a vast array of proteins is a molecular ballet involving intricate steps of transcription, splicing, translation, and post-translational modifications. The resulting diversity not only fuels the complexity of life but also forms the basis for advancements in diagnostic and therapeutic applications, particularly in the fields of IHC, IF, and RNA-FISH. This symphony of techniques harmoniously contributes to the crescendo of molecular biology and medicine knowledge.

As a leader in IHC and IF imaging, Visikol provides clients with high-quality data about protein expression within various tissue types. Through a combination of skilled professionals and cutting-edge technologies, Visikol is pushing the boundaries of immunolabeling and imaging, bringing us closer to a deeper understanding of human health and disease. Reach out to a member of our team today to find out more!

Fluorescence in situ hybridization (FISH) is a molecular technique used to detect and localize specific DNA or RNA sequences within cells, tissues, or whole mount organisms. This technique is based on the use of fluorophore-labeled probes that hybridize complementary DNA or RNA target sequences, allowing for the visualization and identification of specific genetic loci or transcripts. FISH has become an essential tool for both research and diagnostic applications, allowing for the identification of genetic aberrations and disease-associated biomarkers in a variety of tissues and cell types.

FISH Tissue Labeling

The process of FISH tissue labeling with a fluorescent probe involves several key steps. The first step is the selection and design of the appropriate probe sequence. The probe is typically a short, single-stranded DNA or RNA molecule that is complementary to the target sequence of interest. For FISH, the probe is labeled with a fluorophore allowing for its detection and visualization under a fluorescence microscope. The selection of the probe sequence is critical, as it determines the specificity and sensitivity of the FISH assay.

Once the probe sequence has been selected, the next step is to prepare the tissue sample for labeling. This involves fixing the tissue in a solution of formaldehyde, paraformaldehyde, or other fixatives, which preserve the tissue morphology and prevent the degradation of the nucleic acids within the sample. The fixed tissue is then permeabilized with proteases to allow the probe to penetrate the cell membrane and hybridize to the target sequence. Protease treatment also increases hybridization efficiency and reduces background fluorescence of the sample. In the case of whole mount FISH, the organism may need to be cleared, or made transparent, prior to fixation by using clearing agents meant for large tissues, such as Visikol® HISTO™-1 or HISTO™-2. In addition to these two reagents, Visikol® HISTO™-M can be utilized for cell culture models and tissues less that 1mm thick.

After the tissue has been fixed and permeabilized, the next step is the hybridization of the fluorophore-labeled probe to the target sequence. The probe hybridizes to the complementary target sequence within the tissue, allowing for its detection and localization under a fluorescence microscope. After hybridization, the tissue sample is washed to remove any unbound or nonspecifically bound probes. This step is critical to minimize background fluorescence and increase the signal-to-noise ratio of the FISH assay. The washed tissue sample is then ready for imaging using a fluorescence microscope.

FISH and Immunofluorescence

Immunofluorescence imaging is a complementary technique that can be used in conjunction with FISH to visualize specific proteins or other cellular structures within the tissue sample. This technique involves the use of antibodies that are labeled with fluorophores, which recognize and bind to specific protein antigens within the tissue. The tissue sample is incubated with the labeled antibody, allowing for the specific detection and localization of the protein of interest. Both techniques can utilize multiple fluorophores to create multiplex labeling panels to visualize multiple target sequences at once. Here at Visikol, we specialize in highly multiplexed immunofluorescence imaging techniques involving 10+ different antibodies, made possible by our proprietary EasyPlex™ antibody stripping reagent and co-registration software.

Fluorescence in situ hybridization (FISH) is a powerful technique for the visualization and identification of specific genetic loci or transcripts within cells or tissues. The process of tissue labeling with a fluorescent probe involves several key steps, including the selection and design of the appropriate probe sequence, tissue fixation and permeabilization, probe hybridization, washing, and imaging. Immunofluorescence imaging can be used in conjunction with FISH to visualize specific proteins or other cellular structures within the tissue. The combination of these techniques has a wide range of applications in both research and clinical settings, allowing for the identification of disease-associated biomarkers and rapid diagnosis and treatment.

For more information on Visikol’s HISTO™ clearing reagents or our multiplex immunohistochemistry services, please reach out to a member of our team.

References

Egger D, Troxler M, Bienz K. 1994. Light and electron microscopic in situ hybridization: non-radioactive labeling and detection, double hybridization, and combined hybridization–immunocytochemistry. J Histochem Cytochem 42:815–822.

Jin L, Lloyd RV. 1997. In situ hybridization: methods and applications. J Clin Lab Anal 11:2–9.

Rautenstraub BW, Liehr T. 2002. FISH Technology. Berlin: Springer-Verlag.

Hampton, NJ – Visikol has announced that it is partnering with Molecular Instruments (MI) to offer HCR-based RNA FISH and IHC multiplex tissue imaging as a service, adding to the already robust multiplex imaging offering. Visikol provides pharmaceutical and biotech companies with advanced tissue imaging and advanced cell culture assays as a service and today counts all twenty of the top pharmaceutical companies as clients. This addition marks another expansion of the company’s advanced tissue imaging portfolio and aligns with Visikol’s mission of transforming tissues into actionable and quantitative insights for its clients.

“Combining the spatiotemporal patterns of gene expression with protein expression allows Visikol to provide an even greater deal of insights from a single slide, which is incredibly important for precious clinical and preclinical samples,” described Visikol CEO Dr. Michael Johnson.

This new service has been piloted with numerous clients over the last few months and is now available to all Visikol’s clients as a standalone service, or in combination with Visikol’s in-house multiplex IHC approach. The data from the service will also be shared with customers through Visikol’s BitSlide™ platform and can be analyzed using the company’s extensive suite of digital pathology software. “The HCR-based approach for imaging gene expression is unique in its ease-of-use, as well as in its compatibility with multiplex IHC on the same slide. We have also been really impressed by its ability to be used in whole mount thick tissues and are very excited to start offering the technology through our advanced tissue imaging services,” described Visikol CSO Dr. Tom Villani.

Going forward, Visikol looks to continue to work with MI on offering new services that take advantage of novel HCR-based products and is actively seeking to push the envelope on advanced tissue imaging. “HCR amplification technology is appreciated as one of the most significant innovations in bioimaging in several decades, as shown by our users’ extensive publication record working across diverse targets and sample types including whole-mount model/non-model organisms and thin/thick tissue sections. As adoption of HCR-based imaging products grows, we are excited to offer Visikol’s expertise to our users going forward,” commented MI Head of Commercial Dr. Aneesh Acharya.

To learn more about this service click here.

For further information, please contact:
Michael Johnson, CEO, Visikol
Email: Michael.johnson@visikol.com

This information was submitted for publication, through the agency of the contact persons set out above,
on March 11, 2022, at 14:00am (CET).

About Visikol
Visikol is a contract research services company that is focused on accelerating the drug discovery and development process through providing its clients with advanced tissue imaging and advanced cell culture services. Today, Visikol counts all twenty of the top twenty pharmaceutical companies as clients and has been instrumental in dozens of drug discovery programs. The company provides end-to-end services that include 3D tissue imaging, multiplex tissue imaging, digital pathology, high content imaging, 2D cell culture assays, 3D cell culture assays and ex vivo tissue slice assays. Visikol’s expertise lies in both transforming tissues into actionable insights as well as bridging the gap between in vitro assays and in vivo results through the use of bestin-class cell culture models. In addition to its services, Visikol sells a suite of tissue clearing reagents and kits as well as the HUREL® Micro Liver portfolio of primary hepatocyte liver models.

About Molecular Instruments

Molecular Instruments (MI) has the exclusive worldwide license to hybridization chain reaction (HCR) and is the sole developer of HCR-based imaging technologies and reagents. The HCR imaging platform ushers in a new era in which the building blocks of life are quantified at high resolution in the anatomical context of intact tissues. HCR RNA fluorescence in situ hybridization (HCR RNA-FISH) and immunohistochemistry (HCR IHC), enables simultaneous multiplexed, quantitative, high-resolution RNA and protein imaging in the anatomical context of intact tissues, with 1-step, isothermal, enzyme-free HCR signal amplification for all targets simultaneously. The MI team designs, manufactures, and ships reagents from its Los Angeles, CA headquarter to thousands of users around the world across academia, drug development, and diagnostics. The company is proud to stand resolutely by its mantra – “by scientists, for scientists.”