Immunostaining | An Overview of Paraffin Embedded Tissue

Immunohistochemistry (IHC) is a molecular examine that involves the use of antibodies to detect specific proteins within tissues on microscope slides.   These proteins are visualized with a colored chromogen and viewed with a Bright field microscope.  IHC can be used to detect a single protein in tissues or antibodies can be multiplexed to detect two or more proteins on one slide.  Multiplexing is a brilliant way to analysis how proteins are cooperating together within the paraffin embedded tissue or to view several different cell populations at once.

The first and most important step in any histology procedures, including IHC, is fixation. Typically, tissues are fixed in 10% Neutral Buffered Formalin (NBF), processed and paraffin embedding; formalin-fixed, paraffin-embedded tissue. The length of time tissues are in fixative isn’t critical for routine H&E and special staining procedures; however, it is critical for IHC.  Formalin causes cross-linking within the proteins of tissues; these cross-links can mask the epitopes which need to be available to bind to antibodies in IHC procedures.  Over-fixation can render the tissues unusable for IHC.  Ideally, paraffin embedded tissues for IHC should be fixed for 24-48 hours, and then transferred to 70% ethanol until processing, paraffin embedding and sectioning.

Although over-fixation of paraffin embedded tissues can result in un-breakable cross-linking within proteins in tissue, optimal formalin fixation will also form cross-links.  These cross-links can be removed with a procedure called antigen (epitope) retrieval. The two most common methods of antigen retrieval involve the use of heat or enzymes.  Heat-induced epitope retrieval (HIER) involves superheating the slides to temperatures of 90°C-125°C for up to an hour.  HIER is done by placing deparaffinized and hydrated slides are placed in a buffered solution which is then heated up in a pressure cooker, microwave or steamer.  Enzyme-induced epitope retrieval (EIER) is generally performed on hydrated paraffin embedded tissue sections at either room temperature or at 37°C for 5-30 minutes.

Once retrieval procedures are completed, the tissues are blocked for endogenous tissue elements which can interfere with staining or cause background staining.  These endogenous elements can include: proteins, avidin, biotin and peroxidase.  Once blocking is completed, the antibody is applied to the tissue.

Next, antibodies are applied to the paraffin embedded tissue sections. Irrelevant control antibodies of the same immunoglobulin isotype as the primary antibody are applied to a parallel set of slides. For example, if the primary antibody is a Mouse anti-CD3, isotype IgG2a; the irrelevant control antibody is Mouse IgG2a.  The irrelevant control antibody is applied at the same concentration as the primary antibody. The isotype control antibody controls for non-specific binding of antibody to the test tissue. The antibodies are incubated on the slides for 30-120 minutes at room temperature, or overnight at 2-8°C.

The next step is antibody detection.  Detection is the step in which the antibody is bound to a reagent that will react with a chromogen.  The detection is usually done with a secondary antibody that is labeled with an HRP (horseradish peroxidase) polymer.  If the primary antibody is a mouse, than the detection step could be a Goat anti-Mouse HRP polymer.  The polymer is generally applied to the slide for 30-60 minutes.

After the detection step, the colored chromogen is added, and the slides are counter stained.  The most common substrate chromogen is DAB (diaminobenzidine), which produces a brown color when catalyzed by HRP.  Thus, the protein or cell of interest will appear brown on the slide.  Although brown DAB is the most common, there are other colors available; such as purple, pink/red, blue, green and black.  The slides are then counter stained with Hematoxylin, which stains the nuclei a blueish-purple.  Slides are then dehydrated in ethanol, cleared in xylene and cover slipped with resinous mounting medium.

If you want to know more about paraffin embedding, visit our site immunostaining. Here, our team of medical experts will give you all brief history.

Overview of Immunohistochemistry(IHC)

IHC stands for immunhistochemistry. It is a procedure to detect haptens or antigens in cells of a tissues section by using the principal of antibodies uniting particularly to antigens in biological tissues. The antigen-antibody fastening can be imagined in various manners. There are Enzymes like HRP(Horseradish peroxidase) AP( Alkaline Phosphatease) are generally used to catalyze a color making reaction.

It is broadly used in various clinical laboratories and study because this method made it potential to imagine the allotment and localization of particular cellular mechanism within cells and in an appropriate tissue situation. There are various IHC procedures that can be utilized to restrict antigens. The procedure chosen should consist of deliberation of parameters like the specimen kinds and assay sensitivity. Let’s further move to steps that perform immunohistochemistry.

IHC principle and fixation is acquired with a chain of sample preparations that permits manipulation of the tissue for antigen detection. Tissue is surgically placed and removed into a fixative like PFA or NBF. Tissue experiences during IHC fixation where nucleic acids and amines outline semi-reversible cross links collected of methylene bridges. The cross-links make tissue and cell stability and prevent the tissue from damage and decomposition from proteolytic enzymes. Fixed tissues can be stored in paraffin allowing them to be used in future time for IHC.

Samples that are fixed experience a procedure of antigen retrieval to find antigens. Tissues must be rehydrated to allow antibody-apitope connections after mounting and sectioning on slides. Heat-induced epitope retrieval (HIER) is the most common epitope retrieval procedure that uses a mixture of temperature, pH and time to smoothly eradicate cross bridges and expose the antigens of interest.

• Blocking- Reactive sites on proteins are not proteins of interest and endogenous peroxidases can make a fake positive signal. To avoid this, multiple blocking steps are utilized to avoid non-specific binding.

• Detection- Antibody-mediated detection is obtained with fluorescence or chromogenic means. Direct fluorescence detection engages the use of a primary antibody conjugated to a fluorophore. To detect antigens that are not lavishly expressed, indirect fluorescence detection intensifies the signal by using a fluorophore-conjugated secondary antibody. Chromogen detection is made possible with enzyme-conjugated secondary antibodies and a substrate that forms a colored precipitate on the tissue.

• Counterstain – These chemical stains are used to help identify cellular features as well as produce a contrast between the positive antibody Immunohistochemical staining and the universal organelle staining.
• Imaging – Microscopy is done for qualitative analysis, data comparison, and basic visualization of the antigen. We exploit the Aperio VERSA scanner to let for better detail and fine discovery of antigens that are uttered at very low levels. 

IHC has proven to be a priceless examine in the areas of research and drug invention. We stay up to date on the most valuable techniques, detection, and analysis to best meet your needs. If you would like to learn more about how we can help support your study through Immunohistochemistry, visit us at immunostaining.info so that you can know better about it.