Paraffin embedded specimens are cut and mounted on superfrost plus slides. Formalinfixed, paraffin embedded ffpe tissue sections must be treated to remove the paraffin deparaffinization, dewaxing and unmask hidden or latent epitopes in preparation for immunohistochemical ihc staining. Protocol for the preparation and fluorescent ihc staining of paraffin. Immunohistochemistry and staining of ffpe tissues biorad. In both ihc frozen and ihc paraffin experiments the tissue or sections of the tissue are mounted. This article describes an ihc protocol optimized by scientists at cell signaling technology, for use with our antibodies, that you can replicate to obtain the best results in your experiments. Immunohistochemistry ihc detection and identification. The agreement between direct culture and ihc staining was found to be substantial. May 05, 2016 paraffinembedded tissue fragment suspension petfs.
Immunohistochemistry ihc is the most common application of immunostaining. Pathogens in formalinfixed, paraffin embedded sections. Paraffin sections are more physically stable and superior to frozen sections in maintaining tissue morphology with less damage. Transfer the sections onto a poly1lysine coated slide. Fix freshly dissected tissue antibodies, it is recommended to incubate overnight at 28 c. Paraffin removal and antigen retrieval thermo fisher. Handbook of immunohistochemistry and in situ hybridization of human.
Detection of p21 in human lung colon carcinoma by ihc. Paraffin embedding an overview sciencedirect topics. Immunohistochemistry ihc handbook novus biologicals. Depending on how the tissue has been prepared, the ihc method is known as ihc frozen tissue has been snap frozen in liquid nitrogen, isopentane or dry ice or ihc paraffin tissue has been fixed in formaldehyde and embedded in paraffin wax. Multiplex immunohistochemistry for image analysis of tertiary. Compared to paraffin embedded sections, frozen tissues are thicker, lowering microscopic resolution and the ability to capture tissue morphology in detail. Ihc protocol for performing heat hier or proteolytic antigen retrieval pier of paraffin embedded tissue sections. Transfer the sections onto glass slides suitable for immunohistochemistry e. To determine if your sample contains endogenous peroxidase, read more about blocking nonspecific binding.
Epsteinbarr virus ebv epsteinbarr virus ebv latent membrane protein 1 lmp1 is an igg1, kappa monoclonal antibody consisting of commercially individual clones cs1, cs2. Preparation and chromogenic ihc staining of paraffin embedded tissue sections. In order to obtain thin sections for a good ihc result, the fixed sample needs to be embedded in a suitable material providing the necessary hardness to mechanically support the cutting. The results of the present study indicate that the ihc assay is highly sensitive and specific in detecting e. Sections are washed with ddh 2 o before being boiled in antigen retrieval buffer 10 mm trishcl, ph 9. Complete details on how to prepare paraffin embedded cell pellets from both adherent and suspension cells can be found in our cell pellet preparation protocol on page 12. Immunohistochemistry protocol for paraffin embedded tissue. Ma119271 as the primary antibody and an antimouse igghrp conjugate as the secondary antibody. Development and validation of an immunohistochemical. Tissue sections may be dried by onto microscope slides and stored for extended periods at room temperature. Temperature should not exceed 60oc so as to not damage target antigens.
Incubate sections with the primary antibody diluted in 1x tbs for 1. Deparaffinize slides in 2 changes of xylene, 5 min each. Because paraffin is not soluble in water, the tissue has to undergo after. Can you suggest any protocol for immunohistochemistry of paraffin. These immunohistochemical studies employing lowspecificity and highsensitivity. Protocol for the preparation and chromogenic ihc staining. Advantages of ffpe section ihc include excellent tissue morphology and ease of incorporation into routine tissue collection, processing, and storage. Chromogenic ihc staining of paraffin embedded tissue section. Proper slide drying for paraffinsectioned slides labce. This incubation regime allows for optimal specific binding of antibodies to tissue targets and reduces nonspecific background staining. Paraffin is typically heated to 60c and then allowed to harden overnight.
Antigen retrieval an overview sciencedirect topics. Slides are placed in plastic vertical slide holders. Lowspecificity and highsensitivity immunostaining for. Creating great paraffin sections using a rotary microtome takes a great deal of skill and experience. Frozen sections are ideal for quick processing and situations where the antibody may not be compatible with paraffin compounds. To block endogenous peroxidase activity, quench the tissue sections with 3.
The basic steps of the ihc p protocol are as follows. After deparaffinizing and rehydrating the tissue sections, the slides are immersed in an aqueous solution commonly referred to as a retrieval solution. Paraffin embedded tissue paraffin embedding offers the best option for longterm preservation of tissue samples. Argentaffin granules are found in carcinoid tumors. For immunohistochemistry, frozen tissue sections are still the best. Five major steps of paraffin embedding immunostaining. Multilabel immunofluorescence and antigen reprobing on formalin.
Finally, the tissue is sectioned using a microtome. Whichever tissue preparation you decide to go ahead with, please ensure a smooth and uniform sectioning of your tissue using either a cryostat for frozen tissue or a microtome for formalinfixed paraffin embedded tissue. In ihc most commonly used fixative like formalin mask tissue antigens cellular, membrane and nuclear by crosslinking process, this results in poor or no staining in ihc. Incubate the tissue in a 65 c paraffin bath for 2 times, 30 min each. It is not recommended to fix tissue for more than 24. Standardization of immunohistochemistry for formalinfixed, paraffin embedded tissue sections based on the antigenretrieval technique. When we generating paraffin embedded tissue sample, the tissue should be fixed before embedding in paraffin. Detection systems in immunohistochemistry intechopen. Paraffin is the cheapest and most commonly used substance for tissue embedding. Tissue sections are then rehydrated prior to commencing the immunostaining protocol. To overcome this limitation, we have developed a simple and reproducible method to generate apm componentspecific mab which stain formalinfixed, paraffin embedded tissue sections. In most cases, this is achieved with paraffin embedding on formalinfixed material.
The most common controls are cut from the formalinfixed, paraffin embedded tissue. Immunohistochemistry protocol for paraffinembedded tissue. This buffer is intended for heatinduced antigen retriever on formalinfixed paraffin embedded ffpe tissue sections prior to application of antibodies. This kit is designed to stain formalin fixed, paraffin embedded tissue sections, and may be use for frozen sections as well. Immunostaining provides the basic guide for fixation, microtome sectioning and staining of paraffin embedded tissue sections.
Section paraffin blocks at the desired thickness usually 45 m on a microtome and float on a 40c water bath containing distilled water. Immunohistochemistry protocol for paraffinembedded sections. Fix freshly dissected tissue immunohistochemistry ihc with paraffin sections, using variety of antibodies and stains to reveal the molecular architecture of the. The latter are the substrate of choice in immunohistochemical ihc reactions. Protocol for chromogenic staining of paraffin embedded. Preparation and fluorescent ihc staining of paraffin embedded tissue sections. A powerful novel microscope technique is reflection contrast microscopy. Paraffin embedding is perhaps the most commonly used embedding technique. After that i separated the pancreas and cut it into 0. Here is how i did ihc for rats pancreas successfully. Immunohistochemistry protocol for formalin fixed paraffin embedded tissue cut sections at 3 microns and bake overnight at 60c day 1 1. Preparation of paraffin sections for immunohistochemistry sectioning protocol 1. Slides are heated for 20 minutes at 5060oc in a dry oven to facilitate attachment of tissue and to soften the paraffin note.
Mif on formalinfixed paraffin embedded tissue sections. Paraffin embedding paraffin embedding is a method of tissue preservation for immunohistochemistry. Aug 24, 2018 multiplex immunohistochemistry allows the demonstration of multiple protein antigens in individual histological sections of formalinfixed paraffin embedded tumors or other types of tissue. Allow the slides to dry overnight and store slides at room temperature until ready for use. The paraffin tissue block can be stored at room temperature for years. Fluorescent ihc staining of frozen tissue novus biologicals. A method to generate antigenspecific mab capable of staining.
The retriever for formalinfixed, paraffin embedded tissues. Formalinfixed, paraffin embedded ffpe tissue sections must be treated to remove the paraffin deparaffinization, dewaxing and unmask hidden or latent epitopes in preparation for immunohistochemical ihc. Ihc p refers to the staining of tissues that have been fixed usually in neutral buffered formalin and then embedded in paraffin before being sectioned. Immunohistochemistry ihc protocolparaffin section protocol. Jun 04, 2018 the whole process can be performed in nearly 7 min for frozen sections and to less than 3 h for regularly processed, paraffin embedded specimens. This ihc protocol provides a basic guide for the fixation, microtome sectioning, and staining of paraffin embedded. Your fixative solution may influence either negatively or positively the quality of your staining. Preparation and fluorescent ihc staining of paraffinembedded. Read our protocol for the preparation and fluorescent ihc staining of paraffin embedded tissue sections to help with your experiment. Prepare sections of paraffinembedded tissues of no more than. Bake slides at 60c for 30 minutes prior to starting ihc 2. Ihc for paraffin embedded sections video protocol youtube.
The authors preferred proteolytic enzyme used to pretreat formalinfixed, paraffin embedded ffpe tissue sections is 0. Standardization of immunohistochemistry for formalinfixed. Immunohistochemistry ihc detection and identification of. Fixation can be achieved by perfusion or immersion immediately following dissection, and typically requires 424 hours. Immunohistochemistry ihc of formalin fixed paraffinembedded ffpe samples is a very useful tool for examination of macrophages. Section the paraffinembedded tissue block at 58 m thickness on a microtome and float in a 40c water bath containing distilled water. Sections paraffin embedded sections fixations for immunoelectronmicroscopy references bibliography 18 antigen retrieval principal and technique mechanism of action cytology target fpr in situ hybridization antigen retrieval and its use in doublestaining conclusion references 23 staining methods. An overview of ihc staining procedures for formalinfixed. D20 antibody has been used on paraffin embedded tissue sections in a few studies. Preparation and chromogenic ihc staining of paraffinembedded.
Antigen retrieval protocol formalinfixed paraffinembedded. It is a brownblack pigment present normally in the hair, skin, retina, iris, and certain parts of cns. Paraffin sections are deparaffinized in xylene for 2. View all our video protocols with written procedures at. A novel method for quality control preparation in immunohistochemistry. All of the essential aspects of cutting paraffin sections are covered, including.
Deparaffinize slides in xylenes for 5min each and rehydrate through graded alcohols 100% 70% etoh for 5 min each 3. Tissue must be fixed before being embedded in paraffin. Protocol for the preparation and fluorescent ihc staining. It involves the process of selectively identifying antigens proteins in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. Paraffin embedded tissue section is normally sliced by a rotary microtome to give a thickness of 27. Optimum immunohistochemical procedures for analysis of. Immunohistochemistry ihc guide and troubleshooting md.
Immunohistochemistry protocol for formalin fixed paraffin. Immunofluorescence staining of paraffin sections step by step. I am running immunohistochemistry on paraffin embedded slides. However, tissue can also be embedded in plastic, which sets harder and allows thinner tissue sections 1. Paraffin sectioning is the procedure of cutting thin slices of tissue that has been dehydrated and infiltrated with wax using specialized equipment. Keep record of the orientation and sequence of the sections. An overview of ihc staining procedures for formalinfixed, paraffinembedded ffpe tissues contract research organizations cros april 11, 2018 immunohistochemistry ihc is a molecular assay that involves the use of antibodies to detect specific proteins within tissues on microscope slides. Set up negative controls by omitting the primary antibody incubation step for one slide per each experimental condition. Tissue is prepared and preserved through paraffin embedding or cryopreservation freezing.
The second type and most common of embedding media is paraffin. Tissue sections must be rehydrated before commencing the ihc icc protocol. Ihc protocol video for paraffin embedded tissue sections from cell signaling technology cst cst protocols. Fixation time prior to paraffin embedding is generally limited to 2448 hours, which greatly contributes to preservation of antigen integrity. Often the preservation method is closely associated with the type of fixation. This tissue is then embedded in wax before being cut on a microtome. Taylor, in pathobiology of human disease, 2014 abstract. May 12, 2015 the principle of antigen retrieval relies on the application of heat for varying lengths of time to formalin fixed paraffin embedded ffpe tissue sections in an aqueous medium. Comparison of different methodologies and cryostat versus.
The antigen retrieval ar technique of boiling formalinfixed, paraffin embedded ffpe tissue sections in water is a highly effective method to render immunohistochemical ihc staining on ffpe tissue sections, which is a revolution in pathology and a milestone in ihc. Immunohistochemistry ihc is a technique used to analyze protein expression in the context of tissue morphology. Overview of immunohistochemistry ihc thermo fisher scientific. The following protocol mainly describes ihc on paraffin embedded tissues. Ihc staining for p21 in a formalin fixed paraffin embedded ffpe section of human colon carcinoma using a monoclonal antibody cat. A method for fixing and paraffin embedding tissue to. Development of the antigen retrieval ar technique, a simple method of boiling archival paraffin embedded tissue sections in water to enhance the signal of immunohistochemistry ihc, was the fruit of pioneering efforts guided by the philosophy of rendering ihc applicable to routine formalinfixed, paraffin embedded tissues for wide application of ihc in research and clinical pathology. Fix freshly dissected tissue paraffin embedded ffpe tissue sections in water is a highly effective method to render immunohistochemical ihc staining on ffpe tissue sections, which is a revolution in pathology and a milestone in ihc. Frozen sections are typically used for direct or indirect immunofluorescence detection methods and sectioned at a thickness of 10 microns. Immunohistochemical staining of formalin fixed paraffin. Immunostain formalinfixed, paraffin embedded tissue sections steps 929.
Many protocols can be found in the literature and the websites of. Ding w 1, huang k, lu b, xu l, liu j, jiang c, teng x, ren x, wang b. Slides can be stored between 20c and 4c in a dark slide box or slide book. Choosing your fixation embedding and antigen retrieval.
Prepare formalinfixed, paraffin embedded tissue sections step 18. Choosing your fixation, embedding, and antigen retrieval. Preparation and fluorescent ihc staining of paraffin. With proper treatment, the section reveals clear tissue structure and exact antigen location to enable high medicalvalue pathology researches and retrospective studies. Paraffin wax is the most commonly used embedding medium for routine histological applications, and formalinfixed, paraffin embedded ffpe sections produce satisfactory results for detecting most tissue antigens using standard antigen retrieval techniques. The brown precipitating hrp substrate dab was used.
Hence, when a quick and reproducible ihc based diagnostic approach is demanded in emergency circumstances, for example, during surgeries, this method should be taken into consideration 82. Prepare formalinfixed, paraffinembedded tissue sections step 1 8. Preparation and chromogenic ihc staining of paraffin. I perfused the rat first with 100 ml pbs and the then 100 ml 4% pfa. Another potential drawback to frozen tissue is the thickness of the section.
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