Immunohistochemistry (IHC) Basics in Histology (Online CE Course)

(based on 161 customer ratings)

Authors: Carla Shoffeitt, MSM, HT(ASCP) and Diane L. Sterchi, MS, HTL(ASCP)
Reviewer: Donna M. Hedger, BS, HT(ASCP)

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This course introduces the various techniques that are used in the preparation and evaluation of immunohistochemistry (IHC) slides. Procedures and terminology related to IHC are also discussed and strategies for troubleshooting problems are presented.

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Continuing Education Credits

P.A.C.E.® Contact Hours (acceptable for AMT, ASCP, and state recertification): 2.5 hour(s)
Approved through 11/30/2024
Florida Board of Clinical Laboratory Personnel Credit Hours - Immunohistochemistry/Advanced Histologic Techniques: 2.5 hour(s)
Approved through 11/30/2024


  • Explain various immunohistochemical (IHC) techniques used in the laboratory.
  • Identify appropriate quality control (QC) used while performing IHC procedures.
  • Describe slide preparation procedures used in IHC procedures.
  • Define epitope unmasking and retrieval procedures that may be used in performing IHC procedures.
  • Identify and solve problems that can occur when performing IHC procedures.

Customer Ratings

(based on 161 customer ratings)

Course Outline

Click on the links below to preview selected pages from this course.
  • The Role of Immunohistochemistry (IHC) in the Histology Laboratory
  • Antibodies
      • Monoclonal Antibodies
      • Monoclonal antibodies are usually produced in which animal?
      • Which of the following statements are TRUE of a monoclonal antibody? (Select all that apply.)
      • Polyclonal Antibodies
      • Polyclonal antibodies are usually produced in which animals? (Select all that apply).
      • What are the benefits of using a polyclonal antibody? (Select all that apply)
      • Antibody Titering
      • What is the purpose of performing an antibody dilution panel?
      • What range of dilutions would you use for the first dilution panel if the antibody manufacturer suggested a 1:50 dilution?
  • Fixation
      • Tissue Handling
      • Aldehyde Fixation
      • Which of the following is most frequently used in tissue fixation for IHC staining procedures?
      • In regards to IHC staining procedures, which of the following might occur as a result of using 10% Neutral Buffered Formalin as a fixative? (Choose al...
      • Formalin/Aldehyde Substitutes
      • What are some disadvantages of using formalin substitutes? (Choose all that apply.)
  • Processing and Microtomy
      • Tissue Processing Principles
      • Which of the following is TRUE when handling tissue prior to IHC staining?
      • Frozen Sections
      • IHC staining of frozen sections does not require separate validation.
  • Epitope Retrieval
      • General Epitope/Antigen Unmasking
      • Performing Epitope/Antigen Unmasking
      • Which of the following are two types of epitope retrieval most frequently used in IHC?
      • Under what condition is it necessary to unmask antigens?
      • What are the strong bonds called that form between proteins and calcium ions as a result of formalin fixation?
      • At which point in the IHC staining process is epitope unmasking performed?
  • Unmasking Methods
      • Heat-Induced Epitope Retrieval (HIER)
      • What type of heat sources can be used with antigen unmasking?
      • In addition to temperature, which of the following is a very important factor to consider when performing heat induced epitope retrieval procedures?
      • Proteolytic-Induced Epitope Retrieval (PIER)
      • What type of epitope retieval is proteolytic-induced epitope retrieval (PIER)?
      • What two major factors require stringent control when performing proteolytic-induced epitope retrieval (PIER) antigen unmasking?
  • Immunohistochemical (IHC) Methods
      • Immunohistochemical (IHC) Techniques
      • Direct Method
      • The direct method is a multi-step method that uses a labeled antibody that reacts directly with a series of antigens in tissue sections.
      • Indirect Method
      • Which of the following statements are true regarding the indirect method of IHC staining?
      • Peroxidase, Anti-Peroxidase (PAP) Method
      • What does PAP stand for?
      • Which of the following statements is true in regard to the PAP complex?
      • Avidin-Biotin Complex (ABC) Methods
      • Which of the following is considered to be a disadvantage of the avidin-biotin complex (ABC) staining method?
      • The avidin-biotin complex (ABC) method of staining consists of using which of the following? (Choose all that apply.)
      • Labeled Streptavidin-Biotin Complex (LSAB)
      • Polymer-Based Methods
      • What is the advantage of polymer-based IHC methods? Choose all that apply.
  • Blocking Methods
      • Blocking Nonspecific Background Staining
      • Why are blocking methods used as part of IHC staining methods? (Choose all that apply.)
      • Are blocking methods necessary?
      • Avidin-Biotin Blocking
      • Endogenous biotin is often found in what tissues? (Choose all that apply.)
      • When should an avidin-biotin blocking step be performed?
      • Serum Blocking
      • Where in the IHC staining process is a blocking serum utilized?
      • When is a high concentration of blocking serum needed?
      • Blocking Endogenous Peroxidase Activity
      • What does the use of endogenous peroxidase blockers prevent?
      • What reagents may be used to mix the hydrogen peroxide (H2O2) blocking solution? (Choose all that apply.)
      • Blocking Endogenous Alkaline Phosphatase Activity
      • When is it BEST to use an endogenous enzyme blocking method? (Choose all that apply.)
      • What is used to quench high endogenous alkaline phosphatase (AP) activity?
  • Methods of Detection and Visualization
      • Substrate Chromogen Complex
      • What is the primary function of the substrate chromogen complex?
      • Horseradish Peroxidase (HRP)
      • Chromogens Used with HRP
      • Which chromogen-substrate is alcohol and solvent resistant?
      • Alkaline Phosphatase (AP)
      • When using an IHC alkaline phosphatase staining method, what is the mechanism that produces the color?
      • Chromogens Used With Alkaline Phosphatase (AP)
      • Which of the following are common insoluble substrates used with an IHC alkaline phosphatase staining method? (Choose all that apply.)
      • Chromogen Enhancers
      • A blue-black end product color is produced when nickel is added to which of the following substrates?
      • Which of the following can be used as a chromogen enhancer? (Choose all that apply.)
  • Immunohistochemisty (IHC) Staining Reagents
      • Importance of Reagent Integrity
      • What are among the most important factors to consider for all the reagents used in IHC methods? (Choose all that apply.)
      • Antibody Diluents
      • Which of the following are important to consider when preparing antibody diluents? (Choose all that apply.)
      • What hazardous preservation substance is commonly found in antibodies?
      • Buffers and Rinses
      • What role do wash buffers play in IHC staining? (Choose all that apply.)
      • Which of the following reflect the proper storage and handling of wash buffers? (Choose all that apply.)
  • Immunohistochemical (IHC) Controls
      • Quality Control (QC)
      • Why must QC be utilized in the IHC laboratory?
      • Tissue Controls/Control Slides
      • What must be considered when making control blocks?
      • Positive Quality Control (QC) Slides
      • Why is it necessary to use a known positive control?
      • Negative Quality Control (QC) Slides
      • Where is the BEST place to find what negative control to use for any antibody?
      • What is considered the BEST practice for the use of a negative control?
      • System Quality Control (QC)
      • Which antibody is MOST commonly used as a system control?
      • Optimization and Validation of Primary Antibodies
      • What are some important factors to consider when optimizing an antibody? (Select all that apply.)
  • Troubleshooting
      • Troubleshooting
      • Which of the following may be considered the BEST troubleshooting method when staining fails?
  • Record Keeping
      • Record Keeping
      • Why is good record keeping important? (Choose all that apply.)
  • References

Additional Information

Level of instructruction: Basic to intermediate
Intended audience:  This program is designed as an educational and training tool for histology laboratory personnel. This course is also appropriate for students enrolled in clinical laboratory science and histology technician programs as a supplement to classroom instruction and clinical experiences.
Author information: 
Carla J. Shoffeitt, MSM, HT(ASCP) is the Manager of the Pathology Department of Emory St. Joseph’s Hospital of Emory Healthcare in Atlanta, Georgia. She holds a Master of Science Degree in Healthcare Management as well as a Bachelor of Science degree in Biology from Troy University, Troy, Alabama. She is certified as a Histotechnician and has 25 years of experience in the field of Histology.
Diane L. Sterchi, MS, HTL(ASCP) is a senior research associate for Covance Laboratories, Department of Discovery and Translational Services, Pathology in Greenfield, Indiana. She has worked in hospital, veterinary, and research settings in the discipline of histology. Ms. Sterchi holds an MS degree from Indiana Wesleyan University, Marion, Indiana. She has written several textbook chapters and manuals, is a frequent presenter at seminars and has conducted numerous workshops on topics including general histology, immunohistochemistry, and in situ hybridization.
Reviewer information: Donna M. Hedger, BS, HT(ASCP) is a Pathology Manager at Gwinnett Hospital System in Lawrenceville, GA. She graduated from Georgia Southern University in Statesboro, Georgia with a Bachelor of Science degree in biology. She is a certified histotechnician.
Course description:  This course introduces the various techniques that are used in the preparation and evaluation of immunohistochemistry (IHC) slides. Procedures and terminology related to IHC are also discussed and strategies for troubleshooting problems are presented.

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Image provide by Jim Burchette, HT(ASCP)

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IHC Counterstains

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