Ihc staining is commonly used in many research and clinical applications. Rinse the tissue with running tap water for 30min40min to eliminate the formaldehyde. In both ihcfrozen and ihcparaffin experiments the tissue or sections of the tissue are mounted on slides prior to staining. Carry out incubations in a humidified chamber to avoid tissue drying out, which will lead to nonspecific binding and high background staining. Each step of the iccif protocol requires optimization as experimental variables in each step can significantly impact staining outcome. Ihc staining protocol deparaffinization and rehydration protocol permeabilization if staining protocol chromogenic staining protocol ihc workflow troubleshooting guide no or poor signal high background poor tissue morphology uneven or nonspecific staining reference buffers biotechne support products for ihc 1 25 67 89 1011 12 14 1517. Incubate slides with 3% h2o2 solution 100 l for 15 minutes to quench endogenous peroxidase activity.
Steinbusch, free university and mouse antigfp molecular probes. Optimize your experiment with our productspecific protocols for wb, ihc, ip, if, and fc. Protocol for the preparation and chromogenic ihc staining of. Immunohistochemistry ihc basics in histology medialab. We have tested common variations in fixation, permeabilization, and antibody concentration for many of our ifapproved antibodies. For icc experiments, cells must be attached to a microscope slide. To prepare 1 l add 100 ml 10x tbs to 900 ml dh 2 o. Additions and remarks to the methods, made after the apidologie publication. Immunohistochemistry ihc is a technique commonly used for morphological characterization of tumors or other tissue malignancies. Sectioning tissue sections 57 m thick are cut from paraffinembedded blocks on a microtome and mounted from warm water 40c onto adhesive microscope slides.
Achieving reliability and reproducibility of immunostains. Tanisha neely, htascp this course introduces the various techniques that are used in the preparation and evaluation of immunohistochemistry ihc slides. All steps are done with gentle agitation on a rotating platform. As ihc protocol contains many different variable factors, it is challenging to find the best working conditions to.
Ethanol, anhydrous denatured, histological grade 100% and 95% deionized water dh 2 o hematoxylin optional wash buffer. Ihc protocol prestige polyclonals and monoclonals developed for immunohistochemistry based expression profiling are recommended to use according to the standard ihc staining protocol described below. Deparaffinization paraffin sections of 4 m thickness are baked overnight at 50c. Immunohistochemical protocol all ihc staining in the human protein atlas project is performed using a standard protocol as described below. Immunohistochemistry protocol for paraffinembedded tissue.
Education guide immunohistochemical staining methods. Mount tissues should on silane or polyllysine coated slides, or slides coated with histogrip deparaffize slides and, if desired, perform avidinbiotin blocking and endogenous. Procedures and terminology related to ihc are also discussed and strategies for troubleshooting problems are presented. If you would like to contribute your protocols to this site, please go to protocol submission page. Box 488 hudson, wisconsin 540160488 toll free phone. Immunohistochemistry ihc protocolparaffin section protocol. Reagents can be applied manually by pipette, or this protocol can be adapted for automated and semiautomated systems if these are available. Immunohistochemistry protocols thermo fisher scientific us. Immunohistochemistry ihc basics in histology medialab, inc. For the purposes of this guide ihc will be referenced for both ihc and kits. An optimized protocol is necessary to achieve consistent, reliable if results. An antibody that has been shown to work in ihc is preferable. Eisenstat, md 1 primary antibody to dlx2 is m650, dilution 1. Wash sections several times in tbs to remove antifreeze.
Block any nonspecific binding by incubating the tissue sections with 5% animal serum in pbst for 30 minutes at room temperature. The main component of an ihc system is the controller, which is a small computer with inputs and outputs where in or output modules can be connected. In both ihc frozen and ihc paraffin experiments the tissue or sections of the tissue are mounted on slides prior to staining. A simple fivestep selection guide to help you quickly identify and choose the most appropriate reagents for your application. Tube fixation with fixation for 24 hours in 4% formaldehyde, followed by incubation in collagenase for 6. Pdf immunohistochemistry ihc is a favorite tool amongst clinicians to help diagnose a range of diseases by identifying abnormal cells, such as those. Cst aims to help you improve your ihc analysis by providing validated antibodies and reagents to allow you to follow a simple, productspecific protocol and achieve reproducible results with. Education guide immunohistochemical staining methods fifth. Ihcicc protocol guide 4 tissue and cell samples must be appropriately harvested and prepared for each ihcicc study. Prestige antibody immunohistochemistry procedure sigmaaldrich.
Prestige antibodies developed for immunohistochemistry based expression profiling are recommended to be used according to the standard ihc staining protocol described below. You can search by either catalog number or antibody name. Immunohistochemistry ihc is a technique used to analyze protein expression in the context of tissue morphology. To facilitate the required incubation steps, whole tissues must be cut into ultra thin 510 mm sections or cut into smaller pieces for whole mount ihc. Immunohistochemistry ihc is a method for detecting antigens or haptens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. Prepare formalinfixed, paraffinembedded tissue sections step 18. The principles of ihc have been known since the 1930s, but it was not until 1942 that the first ihc study was reported. A shallow plastic box with a sealed lid and wet tissue paper. Fix dissected tissues with 10% formalin for no less than 48 hours at room temperature. This new ihc resource guide will help you navigate through the many choices of detection systems, substrates, and ancillary reagents that complete your workflow. Immunohistochemistry technically, ihc and icc are relatively simple and straightforward experimental methods. Find your productspecific protocols for wb, ihc, ip and more. This section of the guide will highlight some of the data we use to support our if protocol recommendations. This ihc protocol provides a basic guide for the fixation, cryostat sectioning, and staining of frozen tissue samples.
Tissue predigestion tissue digestion recommendations for all predilute and 50x concentrated ihc antibodies are given in our anatomical pathology catalog. Immunohistochemistry ihc is an important application of monoclonal as well as polyclonal antibodies to determine the tissue distribution of an antigen of interest in health and disease. Place slides in a glass slide holder and fill in the rest of the rack with blank. Rinse slides with pbs and start the immunostaining protocol. Ihc staining methods, fifth edition 1 the pivotal reagent common to all immunohistochemical techniques is the antibody. This guide highlights critical steps in the ihc protocol and demonstrates how protocol changes can affect the final. The products supplied by atlas antibodies are warranted to meet stated product specifications and to confirm to label descriptions when used and stored properly.
Immunohistochemistry ihc staining protocol below is an overview of our recommended protocol and highlighted are the key steps to a successful experiment. Protocol for the preparation and chromogenic ihc staining. The species of the animal serum used in permeabilization and blocking buffers is dependent on the host of your secondary antibody. The researcher should optimize the precise conditions for a particular assay. This course introduces the various techniques that are used in the preparation and evaluation of immunohistochemistry ihc slides. Incubate for 10 min utes at room temperature in methanolperoxidase.
Immunohistochemistry overview immunohistochemistry ihc is a method to detect specific target antigens proteins in tissue sections using antibodies. Principles of immunohistochemistry ihc staining cell. Pdf immunohistochemistry protocol and basic information. Technically, ihc and icc are relatively simple and straightforward experimental methods. The most comprehensive ihc guide ever created bosterbio. Determination of humidity by digital refractometry method 1 decided at the dijon meeting in october 1999 determination of invertase method 10. Ihc staining protocol for paraffin, frozen, and free floating sections. If you would like to contribute your protocols to this site, please go to protocol submission page for details or contact us to add your lab protocol links. Immunohistochemistry instruction manual immunostar, inc. Prior use in ihc an antibody that recognizes its target protein in western blotting experiments may not always recognize the antigen in ihc, where the antigen is more likely to be in its native tertiary 3d form. Basic iccif protocol a standard iccif protocol involves fixation, permeabilization, blocking, immunolabeling, counterstaining, and microscopic imaging of stained cells see the flow chart in figure 2. However, there are many variables which must be identified and optimized for each individual ihcicc study. No changes can be made without the approval of the ihc.
The ihc protocol contains many steps that may require optimization to ensure specific antibody binding and optimal visualization of the target protein. 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. 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. Box 488 hudson, wisconsin 540160488 tollfree phone. This protocol database is compiled based on published literatures, laboratory submissions, as well as individual experimental data. Read more about protocol for the preparation and fluorescent ihc staining of frozen tissue sections. 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. Since then, major improvements have been made in tissue fixation and sectioning methods, antigenepitope retrieval, antibody conjugation. Ihc standard protocol optimized for triple a polyclonals and precisa monoclonals from atlas antibodies. This is in contrast to the ihc free floating technique where the entire. In addition, the newer versions of the controller are wireless so you can use various wireless switches, dimmers and relays. Rinse slides with ps for 3 times, 5 minutes each time.
Florida board of clinical laboratory science ce immunohistochemistryadvanced histologic. Inadequately fixation can make tissues dehydrated during tissue processing and become hard and brittle. Read more about protocol for the preparation and fluorescent ihc staining of frozen tissue sections troubleshooting guide. Immunocytochemistry icc uses similar techniques to localize cellular proteins in cell preparations.