Immunoelectron Microscopy Imaging Service
Introduction
Immunoelectron microscopy (IEM) stands as a pivotal technique in the realm of cellular and molecular biology. It amalgamates the high-resolution capabilities of electron microscopy with the specificity of immunolabeling, enabling the precise localization of proteins and other macromolecules within cellular structures. This method is predicated on antigen recognition by primary antibodies, which are subsequently visualized through electron-opaque gold granules, rendering them discernible in transmission electron microscopy (TEM) images.
Fig.1 In a microscopic image, tiny dark circular entities, comprising gold nanoparticles enveloped with a monoclonal antibody targeted at rotavirus protein VP6, adhere to rotaviruses1.
Applications
The applications of IEM are vast and multifaceted. For instance, it has been instrumental in identifying native platelets in aqueous solutions, discerning glutathione contents in different leaf areas of Arabidopsis, and even in the intricate study of the tumor microenvironment from human breast cancer samples. Furthermore, IEM has been adeptly employed to quantify distributions of proteins across cellular compartments, offering invaluable insights into biological responses. Its ability to visualize nanoparticles, especially when antibodies to surface materials are available, underscores its versatility.
Sample Requirements
The key to the successful application of immunoelectron microscopy is:
- Proper preservation of the ultrastructure of cells.
- Ensuring that the antigen of the cell or its subcellular structures remains in place and retains its antigenicity.
- The selected immunoreagents should be able to penetrate the tissue and cell structures and bind to the antigens. This technique is mainly used for the localization of viruses, bacteria, and other antigens, understanding the pathogenesis of immune diseases, and ultrastructural immunocytochemical research, among others.
Specific operating procedures:
1. Take a sample within 1-3 min. The sampled tissue should be 1mm³ in size. Before sampling, prepare a dish with Fixative A for immunoelectron microscopy. Once the tissue chunk is excised, immediately place it in the dish. Using a scalpel, cut the tissue in the fixative into 1mm³ chunks. Then, transfer the cut tissue into an Eppendorf tube containing fresh fixative for continued fixation.
2. When sampling, try to be as precise as possible to the target region (e.g., for observing the glomerulus, sample the renal cortex; for observing the islets, sample the islet-rich tail of the pancreas; for tissues like skin and the gastrointestinal tract that can curl in the fixative, you can affix the tissue onto a filter paper for fixation).
3. During sampling, take care to avoid mechanical damage like squeezing with forceps, and ensure the scalpel is sharp to prevent tissue bruising.
4. After excising the tissue, immediately immerse it in the specialized fixative for immunoelectron microscopy, store at 4°C, and transport with ice packs at 4°C. Ensure the fixative doesn't freeze during storage and transport. After fixing the sample, proceed to embedding and immunolabeling as soon as possible to reduce the possibility of antigen loss.
5. Observing and collecting images: Examine under a transmission electron microscope, and capture images. Black gold particles of 10 nm in size indicate positive expression.
Service Process
1. Discuss the project with the customer and sign the project contract. This initial step ensures that both parties have a clear understanding of the project's objectives and expectations.
2. Sample collection and preparation: The client provides the sample, ensuring it meets the aforementioned requirements.
3. Antibody selection and application: Based on the target antigen, specific primary antibodies are chosen and applied to the sample.
4. Visualization and imaging: Electron-opaque gold granules are introduced, and the sample is imaged using TEM.
5. Data analysis and report generation: Examining the captured visuals, an in-depth analysis ensues, culminating in the creation of an extensive document elucidating the discoveries.
6. Feedback and revisions: The client reviews the report, and any necessary revisions are made based on their feedback.
Creative Biolabs' Immunoelectron Microscopy Imaging Service
Creative Biolabs offers an unparalleled IEM imaging service. With a team of seasoned experts and state-of-the-art equipment, we guarantee precise, high-resolution imaging tailored to the client's specific needs. Our collaborative approach, as exemplified by their rigorous service process, ensures that every project is executed with the utmost precision and professionalism.
For more information, please contact us.
FAQs
Q1: What is the resolution of IEM compared to traditional electron microscopy?
A: IEM offers a similar resolution to traditional electron microscopy but with the added advantage of specific antigen localization.
Q2: How long does the entire IEM process take?
A: The duration varies based on the sample's complexity and the project's specific requirements. However, on average, it can range from a few days to several weeks.
Q3: Are there any limitations to the types of samples that can be used for IEM?
A: While IEM is versatile, the sample's quality and preparation play a pivotal role in the accuracy of the results. It's essential to consult with the service provider regarding any potential limitations.
Q4: How is IEM different from immunofluorescence microscopy?
A: While both techniques are based on immunolabeling, IEM uses electron microscopy for visualization, offering a much higher resolution than the light microscopy used in immunofluorescence.
Reference
- From Wikipedia: https://en.wikipedia.org/wiki/Immune_electron_microscopy#/media/File:Rotavirus_with_gold-_labelled_monoclonal_antibody.jpg
