Transfer the sections onto glass slides suitable for immunohistochemistry e. Ihcp antibodies optimized for staining of paraffin embedded tissues 425805 6fbb3049baa7. Immunofluorescence and immunohistochemistry sciencedirect. Immunohistochemistry technically, ihc and icc are relatively simple and straightforward experimental methods. An enhanced antigenretrieval protocol for immunohistochemical staining of. Repeat this step again in fresh xylene for a further 10 minutes. Protocol for the preparation and fluorescent ihc staining. Incomplete removal of paraffin can lead to poor staining of the section. Though less sensitive quantitatively than immunoassays such as western blotting or elisa, it enables the observation of. Today, the most commonly used methods in immunohistochemistry refer to histological sections obtained from paraffin sections, cryostat or cell suspensions.
Indirect immunostaining protocol for paraffin embedded tissue sections. Fix dissected tissues with 10% formalin for no less than 48 hours at room temperature. Mount sections on to charged slides and dry overnight. Can you suggest any protocol for immunohistochemistry of. Paraffin tissue sections for ihc are usually cut at a thickness of 4 to 6. The basic steps of the ihcp protocol are as follows. This article describes an ihc protocol optimized by scientists at cell signaling technology, for use with our antibodies, that you can replicate to. Wash the slides 3 times with distilled water, 2 minutes each.
Immunohistochemistry protocol for paraffinembedded sections. The first decision when planning an ihc study is how to prepare the tissue sections. All of the essential aspects of cutting paraffin sections are covered, including. Immunohistochemical staining methods dako handbook ihc world. In many cases incubation times will vary depending upon the particular processing method that has been used, or for different antigens. The basics of immunohistochemistry in the kidney we use immunohistochemistry ihc in both a qualitative and a semiquantitative manner, i. Can you suggest any protocol for immunohistochemistry of paraffin.
Following the preparation of a paraffin section, all the elements are infiltrated with and surrounded by paraffin wax which is hydrophobic and impervious to aqueous reagents. Paraffin embedding an overview sciencedirect topics. Primary antibodies tested in ihcparaffin sections biorad. Dilute the primary antibody to the manufacturers recommendations or to a previously optimized dilution. The key feature of this protocol is treatment of deparaffinized tissue sections at. These protocols are the result of extensive inhouse validation performed at cst and ensure accurate and reproducible results product specific protocols will be linked from matching product web pages. Standardization of immunohistochemistry for formalinfixed. Allow the slides to dry overnight and store slides at room temperature until ready for use. Ihc protocol immunohistochemistry protocols pages 1 5. Immunohistochemistry ihc protocolparaffin section protocol.
Paraffin and frozen sections reagents can be applied manually by pipette, or this protocol can be adapted for automated and semiautomated systems if these are available. Photographs of a tissue sample, paraffin block, and resulting tissue. Indirect immunostaining of paraffinembedded tissue sections. Slides can be stored between 20c and 4c in a dark slide box or slide book. Infiltrate and embed in paraffin use automatic processor. Immunohistochemistry protocols thermo fisher scientific us. Alternatively superfrost or polyllysine coated slides can be used. Sample fixation is key to tissue processing and is critical to prevent the degradation of. If deparaffinization is not complete, the target antigens will be obscured and the antibodies will be unable to react with them. Add primary antibody diluted in 1% animal serum pbs with or without 0. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides.
Immunohistochemistry protocol for paraffin embedded. The paraffin in ffpe sections must be completely removed before ihc staining. Add the primary antibody diluted in 1% animal serum in pbst and incubate at room temperature for 12 hours. This guide aims to help you improve your ihc analysis by providing suggestions to allow you to achieve the expected results with minimal enduser optimization. Immunohistochemistry ihc is a technique used to analyze protein expression in the context of tissue morphology. Prepare formalinfixed, paraffinembedded tissue sections step 18. Though less sensitive quantitatively than immunoassays such as western blotting or elisa, it enables. Prepare peroxidase blocking solution by adding one part 30% hydrogen peroxide to nine parts methanol. Transfer the sections onto a poly1lysine coated slide.
In this video, we demonstrate the ihc protocol we have optimized for formalinfixed paraffin embedded ffpe tissue sections so you can replicate results. Most antibodies will be used in ihcp at a concentration of 0. Place the lid loosely on the staining dish and incubate for 2040 minutes optimal incubation time should be determined by user. Standardization of immunohistochemistry for formalinfixed, paraffinembedded tissue sections based on the antigenretrieval technique.
The following steps reflect the protocol for paraffinembedded samples. Immunohistochemistry protocol for paraffin embedded tissue sections duration. Enzos complete set of tools improve the quality and efficiency of your ihc process from start to finish. Immunohistochemistry protocol for paraffinembedded tissue. Creating great paraffin sections using a rotary microtome takes a great deal of skill and experience. Using charged slides helps the section to adhere to the slide. Primary antibody solution see following is gently pipetted onto tissue sections. Ihcp refers to the staining of tissues that have been fixed usually in neutral buffered formalin and then embedded in paraffin before being sectioned. Block each section with 100400 l blocking solution for 1 hour at room temperature. Prepare formalinfixed, paraffinembedded tissue sections step 1 8. We developed a novel modified antigenretrieval protocol for reversing the effect. Ihc is a challenging application and problems often occur. Inadequately fixation can make tissues dehydrated during tissue processing and become hard and brittle.
Ihc deparaffinization protocol this step is required when using paraffin embedded sections. Immunohistochemistry preparation and staining of paraffin sections. Summary workflow chart for ihc protocol for paraffin embedded sections. An enhanced antigenretrieval protocol for immunohistochemical. May be used with frozen or paraffinembedded sections. These staining times can be adjusted as necessary for protocol optimization. Keep record of the orientation and sequence of the sections. Microtomy and paraffin section preparation is a great training aid for new microtomists and is an excellent refresher for experienced operators. Frozen sections and floating sections are other options each method has advantages and limitations table 1. Consult product data sheet for recommended antibody diluent. Immunohistochemistry protocol for paraffin embedded tissue.
Immunohistochemistry ihc solutions enzo life sciences. Here, i outline some tips for each of these steps and highlight which may need a little tweaking. Lanjing zhang and fan lin 2017 best practices in immunohistochemistry in surgical pathology and cytopathology. Materials and reagents xylene 100% ethanol 95% ethanol 70% ethanol 50% ethanol.
Ihc staining protocol contents paraffin and frozen sections immunostaining freefloating sections signal amplification paraffin and frozen sections reagents can be applied manually by pipette, or the protocol can be adapted for automated and semiautomated systems if these are available. Read more about protocol for the preparation and fluorescent ihc staining of frozen tissue sections troubleshooting guide. Ihc staining viewed on a laser scanning confocal microscope. Protocol immunohistochemistry protocol for paraffin. Cryostat sections are picked up on subbed slides chromealum gelatin coated and air dried for at least 2 hours. Block each section with universal blocking buffer for 30 min at rt. The ihc protocol is straightforward, but contains many steps that may require initial optimization to ensure specific antibody binding and optimal visualization of the target protein. Before proceeding with the staining protocol, the slides must be deparaffinized and rehydrated.
Immunohistochemistry is a method of detecting the presence of specific proteins in cells or tissues and consists of the following steps. Drain slides for a few seconds do not rinse and wipe around the sections with tissue paper. Find and buy antibodies tested for your immunohistochemistry ihcp staining experiments. Standard staining procedures the internet pathology laboratory for medical education, florida state university college of medicine standardized staining procedures for histology laboratory.
Continuous sections staining may be the simplest way to visualize two antigens. Harvest fresh tissue and place it in a dish filled with icecold pbs buffer. Histologystaining protocols protocol online your lab. For decades, immunohistochemistry ihc has been an invaluable tool for the detection, localization, and quantification of antigens in preserved tissue for research and diagnostic purposes.
Immunostaining of paraffin sections the open lab book v1. Immunohistochemistry ihc handbook novus biologicals. Preparation of paraffin sections for immunohistochemistry sectioning protocol 1. The recommended thickness of tissue section for ihc is mostly 4. Ihcfrozen, and the sections are then probed with primary antibodies against the antigens of interest. The researcher should optimize the precise conditions for a particular assay. Immunohistochemistry double staining technique can be used to visualize two or more antigens and analyze the relationship of their localization, morphology and functions. Immunofluorescent staining of paraffinembedded tissue. The conventional light microscopy technique involves obtaining the tissue sample, followed by fixation, paraffin embedding, sectioning, mounting on a glass slide, staining, and examination. Incubate sections in 3% hydrogen peroxide for 10 minutes. Rinse the tissue with running tap water for 30min40min to eliminate the formaldehyde.
Specimen processing preparation of tissue sections fixing and dewaxing. Ihc protocol for paraffin embedded sections bosterbio. Before you begin, determine the fixation method \formalin or zinc\ and the method of heating slides \microwave or pressure cooker or autoclave\ for antigen retrieval, if needed. In this protocol, we discuss the preparation of paraffinembedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and nonspecific binding sites. Ihcparaffin protocol ihc p immunohistochemistry or ihc is a method for demonstrating the presence and location of proteins in tissue sections.
Ihc workflow paraffin sections with applicable bosters reagents. Carry out incubations in a humidified chamber to avoid tissue drying out, which will lead to nonspecific binding and high background staining. In fact, paraffins hydrophobicity actually repels aqueous solutions containing the ihc staining reagents. As shown in the name, it respectively stains two antigens. Wash sections in dh 2 o three times for 5 minutes each. Ihc for paraffin embedded sections video protocol duration.
Photographs of conventional tissue sections taken through a light microscope photomicrographs were illustrated in figure ii. For the best possible results, cell signaling technology cst strongly recommends using our optimized applicationspecific protocols for each product. Certain paraffinembedded sections need to have an antigen retrieval procedure to uncover antigenic sites from tissue sections. In spite of its inherent detrimental effects on antigenicity and ihc, a proper and consistent protocol for fixation with neutrally buffered formalin is imperative for highquality ihc. Prepare formalinfixed, paraffinembedded tissue sections steps 18. This ihc protocol provides a basic guide for the fixation, microtome sectioning, and staining of paraffinembedded tissue samples. All steps in the following protocol are carried out at room temperature unless stated otherwise. Immunohistochemistry or ihc is a method for demonstrating the presence and location of proteins in tissue sections. The first thing to consider is the choice of primary antibody. Section the paraffinembedded tissue block at 58 m thickness on a microtome and float in a 40c water bath containing distilled water.