IHC Embedding Optimization

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IHC Optimization Tips Staining at Immunostaining.info

Optimizing IHC Staining is a process in Immunohistochemistry (IHC) which is a critical technique in both clinical and life science research laboratories, combining the power of specific antibodies and microscopy to reveal both the location and abundance of a given protein in a tissue section. Founded in 1993 by famed histologist Steven Xia, Boster Bio is an antibody manufacturer specializing in high-sensitivity, high-specificity, ELISA kits and WB/IHC compatible antibodies. They offer antibodies that are thoroughly validated for numerous applications including IHC, WB, ELISA and Flow Cytometry. Team Boster strives to provide best service and has earned the trust of researchers worldwide. All products are covered by the Boster Quality Guarantee, so each will work as advertised or your money back. Boster product lines are actively expanding with new antibodies and ELISA kits every month.

In theory, the protocol is straightforward in IHC Optimization Tips Staining. A thinly sliced tissue sample, either flash-frozen or formalin-fixed and paraffin-embedded, is placed on a microscope slide and incubated with an antibody specific to the protein of interest followed by signal detection using a chromogenic substrate or fluorescent dye. The signal is observed microscopically in areas where the antibody is bound to the protein of interest.

In reality, IHC Fixation Protocol Staining for an IHC assay involves managing several key parameters and can be labour intensive. While a standard protocol may work very well with some antibodies, the same protocol might not work perfectly with all antibodies, and certain antibodies might require protocol modifications in order to generate optimal results.

Some of the key considerations users must bear in mind when setting up their own IHC experiments is to understand your antibody. Commercial antibodies often come with datasheets that assert the antibody works for IHC and include a colour image of a stained tissue section; however, it is still essential to perform experiments to confirm antibody specificity in your cell type. It is equally important to characterize homemade antibodies rigorously to ensure antibody performance and specificity.

About immunostaining.info

Immunostaining.info is the website that offers you detailed information about IHC protocol. It is a small part of Boster Bio that gives you complete knowledge about IHC principle and IHC protocol. IHC principle is the method of fixation, tissue sectioning, paraffin embedding, inactivation and blocking, antigen retrieval, detection and so on. You will also study complete guide of IHC Sample preparation guide and IHC protocol. To know more about it, visit us at immunostaining.info and get complete information. For more details visit our website https://immunostaining.info/

Address:

Immunohistochemical Staining

3942 Valley Ave

Pleasanton, CA 94566, USA

Email:[email protected]

Phone: [phone]

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IHC Antigen Retrieval Optimization Tips

Find the IHC antigen retrieval optimization tips to select the right antigen retrieval methods that fix a tissue sample with paraformaldehyde causes the formation of peptide crosslinks.

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Discover Antigen Retrieval Methods at Immunostaining.info

Although fixation is essential for the preservation of tissue morphology, this process can also have a negative impact on IHC/ICC detection. Fixation can alter protein biochemistry such that the epitope of interest is masked and can no longer bind to the primary antibody. Masking of the epitope can be caused by cross-linking of amino acids within the epitope, cross-linking unrelated peptides at or near an epitope, altering the conformation of an epitope, or altering the electrostatic charge of the antigen. Antigen Retrieval Methods refers to any technique in which the masking of an epitope is reversed and epitope-antibody binding is restored.

Immunostaining is the website that offers you detailed information about IHC Protocol. It is a small part of Boster bio that gives you complete knowledge about IHC Principle and IHC Protocol. This website is especially designed to educate the people about the detailed process of immunostaining and how it works?

According to the experts at immunostaining.info formaldehyde fixation usually generates methylene bridges which cross-link proteins and therefore mask the epitope of interest. It is essential to unmask the antigen epitopes in order to allow the antibodies to bind, either by heat (Heat Induced Epitope Retrieval: HIER) or enzymatic digestion (Proteolytic Induced Epitope Retrieval: PIER).

The HIER method can be implemented by microwave, high pressure or water bath. It breaks the methylene bridges and exposes the epitope to allow the antibodies to bind by continuously heating. The following antigen retrieval reagent is required:

You can find IHC Antigen Retrieval Optimization Tips at the resource immunostaining.info. While selecting the right antigen retrieval method, the process of fixing a tissue sample with paraformaldehyde causes the formation of peptide crosslinks that serve to preserve tissue architecture and protein localization. These peptide crosslinks can also mask epitopes, making them inaccessible to antibody binding. In order to get good-quality IHC stains, these cross links must be broken in the process known as epitope retrieval.

There are two common methods of epitope retrieval: heat-induced epitope retrieval (HIER), and proteolytic-induced epitope retrieval (PIER). Selecting the right method is critical to get the best results for your application.

About immunostaining.info

Immunostaining.info is the website that offers you detailed information about IHC protocol. It is a small part of Boster Bio that gives you complete knowledge about IHC principle and IHC protocol. IHC principle is the method of fixation, tissue sectioning, paraffin embedding, inactivation and blocking, antigen retrieval, detection and so on. You will also study complete guide of IHC Sample preparation guide and IHC protocol. For more details visit our website https://immunostaining.info

Address:

Immunohistochemical Staining

3942 Valley Ave

Pleasanton, CA 94566, USA

Email:[email protected]

Phone: [phone]

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IHC Mounting Media

Here you can learn more about IHC mounting media used in immunohistochemistry. An IHC mounting medium may be used to attach a coverslip or may itself be used to replace the coverslip.

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Check out IHC Troubleshooting Tips at Immunostaining.Info

Did you know that the immunohistochemistry technique is used in the search for cell or tissue antigens that range from amino acids and proteins to infectious agents and specific cellular populations! The technique comprises two phases: one is - the slides preparation and stages involved for the reaction and second is - interpretation and quantification of the obtained expression. Immunohistochemistry is an important tool for scientific research and also a complementary technique for the elucidation of differential diagnoses which are not determinable by conventional analysis with hematoxylin and eosin.

Boster Bio is one of the leading Flow Cytometry Technical Resource Centers. It was founded by Steven Xia, histologist, in 1993. Their products are enclosed by the Boster Quality Guarantee and vigorously mounting with new antibodies and ELISA kits. The corporation currently provides hybridoma generation check to product custom monoclonal antibodies. They are proficient in ELISA kits and antibody-antigen liquid phase reactions. For IHC troubleshooting tips you can check out immunostaining.info.

This website is especially designed to educate the people about the detailed process of immunostaining and how it works?

“Initially I was working on the website Boster Bio and focused on the treatment with our effective medicines. IHC was something that I have not studied and was keenly interested in doing the research work so that I can do something great for my fellow beings. Here, silver jubilee of Boster Bio was near and the idea struck in my why not to introduce particular website related to immunostaining and the result was immunostaining.info. Here, you will get every kind of information that is related to the tissue sectioning, IHC protocols and many more.”

Immunohistochemistry is an umbrella term that encompasses many methods used to determine tissue constituents (the antigens) with the employment of specific antibodies that can be visualized through staining. When used in cell preparations it is called immunocytochemistry, a term that experts use for all methods entailing the immunological search of cell antigens, even when this involves tissue slices.

In the last couple of decades there has been an exponential increase in immunohistochemistry techniques and thus immunostaining.info provides IHC Troubleshooting Tips Specific Staining.

About immunostaining.info:

Immunostaining.info is the website that offers you detailed information about IHC protocol. It is a small part of Boster Bio that gives you complete knowledge about IHC principle and IHC protocol. IHC principle is the method of fixation, tissue sectioning, paraffin embedding, inactivation and blocking, antigen retrieval, detection and so on. You will also study complete guide of IHC Sample preparation guide and IHC protocol. To know more about IHC Troubleshooting Tips, visit us at https://immunostaining.info/ and get complete information.

Address:

Immunohistochemical Staining

3942 Valley Ave

Pleasanton, CA 94566, USA

Email:[email protected]

Phone: [phone]

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Paraffin Embedding Overview - Immunostaining.info

Paraffin embedding is one of the standard procedures used in preparing slender sections of biological substance for histological assessment by light microscopy. There are major steps that are involved in paraffin embedding includes fixation, dehydration, transparentizing, immersion and embedding.

The tissues samples collected are usually submitted to regular pathologic analysis and then preceded through fixation and paraffin embedding. Fixation procedure is used to cross-link the antigens to well-built macromolecules, thereby immobilizing them in tissue.

Furthermore, paraffin embedding may have an effect on the immunoreactivity. For predictable histology, fixation of tissue, followed by paraffin embedding is the most widely-used method. It is criterion in the majority of histology laboratories, and is appropriate for assessing lymphoid tissue.

Here are the 5 steps described that are involved in paraffin embedding:

Fixation

The main purpose of this process is to keep cell sharp and tissue in proper shape to prevent putridness, postmortem autolysis, endogenic and exogenic enzyme activity. Maintain the cell structure as well as position by preventing antigen diffusion. It gives color to clarify tissues by diverse affinity to coloring agent.

Dehydration

In this step the water is completely removed and creates a situation for the next step and hardens the tissue of curiosity. The dehydrating agents Ethanol and Acetone are completely miscible with water and can be arranged in different volumetric ratios.

Transparentizing

The tissue of interest necessitates a transparentizing move for the reason that the dehydrating agent used in the above step is immiscible with the paraffin. The adding up of transparent reagent helps paraffin soak up into the tissue.

Immersion

When the transparentizing process is completed, the tissue is then immersed in molten paraffin wax so that it takes up the wax-substituting transparent source. Based upon the melting temperature of paraffin wax, immersion should be implemented in between 54℃ to 64℃.

Embedding

In embedding the process is carried of treating the tissue in a paraffin box so that the paraffin wax cools down plus solidifies. Once cooled, the tissue will be ready for sectioning and appropriate for storage.

About Boster Bio

As the leading Flow Cytometry Technical Resource Centers, Boster Bio was founded by Steven Xia, histologist, in the year 1993. The business currently provides hybridoma generation check to product custom monoclonal antibodies. For more information on Paraffin Embedded Tissue, please visit https://immunostaining.info.

Contact Address

Boster Biological Technology

3942 Valley Ave

Pleasanton, CA 94566, USA

Email:[email protected]

Phone: [phone]

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Paraffin Embedded Tissue

Paraffin Embedding involves the steps fixation, dehydration, transparentizing, immersion and embedding. Paraffin Embedded Tissue is sliced by a rotary microtome to give a thickness of 2-7 μm.

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Antigen Retrieval Methods

Find the IHC antigen retrieval optimization tips to select the right antigen retrieval methods that fix a tissue sample with paraformaldehyde causes the formation of peptide crosslinks.

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Discover IHC Mounting Media at Immunostaining.info

Immunostaining is the online resource that offers you detailed information about IHC Protocol. It is a small part of Boster bio that gives you complete knowledge about IHC Principle and IHC Protocol. This website is especially designed to educate the people about the detailed process of immunostaining and how it works?

After staining the target antigen by IHC, a secondary stain is usually applied to provide contrast that helps the primary stain more distinct. While many of these stains show specificity for discrete antigens or cellular compartments, other stains will deliver the staining of a whole cell. This counter stain will change the nucleus to red after applying to the tissue section for 2 to 5 min. IHC mounting media may be used to attach a cover slip or may it be used to replace the cover slip. Generally, the medium selection depends on a few factors including the chemical compatibility with chromogen and counter stain as well as the preservation period.

As it is Neutral Mounting Medium usually refers to an oily substance with pH 7.0 such as neutral gum (resin). Before mounting, the sample should be treated with dimethyl benzene, transparent and dehydrated for long-term storage sections.

Water-Soluble Mounting Medium, popularly used in IF staining for short-term storage sections, this mounting medium usually consists of 50% glycerol.

Once your tissue sample is fixed using the appropriate method and fixative, it is ready for sectioning and mounting. In order to preserve tissue morphology during sectioning, the tissue must be embedded in paraffin. If you are performing IHC Embedding Optimization on frozen sections, fixation occurs after sectioning. The decision to use frozen or formalin fixed paraffin embedded (FFPE) samples is dependent on which downstream applications you intend to use.

Praffin embedded Frozen

Fixation step Pre-embedding Post-sectioning

Sectioning equipment    Microtome Cryostat

Storage stability Years at room temperature One year at -20C

Advantages includes Tissue morphology preservation Enzyme, activity preservation

About immunostaining.info Immunostaining.info is the website that offers you detailed information about IHC protocol. It is a small part of Boster Bio that gives you complete knowledge about IHC principle and IHC protocol. IHC principle is the method of fixation, tissue sectioning, paraffin embedding, inactivation and blocking, antigen retrieval, detection and so on. You will also study complete guide of IHC Sample preparation guide and IHC protocol. For more details visit our website https://immunostaining.info/

Address Immunohistochemical Staining 3942 Valley Ave Pleasanton, CA 94566, USA Email:[email protected] Phone: [phone]

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IHC Embedding Optimization

IHC Embedding Optimization gives you information about the decision to use frozen or formalin fixed paraffin embedded (FFPE) samples is dependent on which downstream applications you intend to use.

For more details about IHC Embedding Optimization, visit our website https://immunostaining.info