Immunohistochemistry (IHC) Basics in Histology (Online Course)

(based on 158 customer ratings)

Authors: Diane L. Sterchi, MS, HTL(ASCP) and Donna M. Hedger, BS, HT(ASCP)
Reviewer: Tanisha Neely, HT(ASCP)

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

Objectives

  • 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 158 customer ratings)

Course Outline

  • The Role of Immunohistochemistry (IHC) in the Histology Laboratory
      • Immunohistochemistry (IHC) Uses and Considerations
      • Antibodies, Antigens, and Epitopes
      • Match each of the following terms to the correct definition.
      • Antibodies may attach to:
  • 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?
      • What are the benefits of using a polyclonal antibody?
      • 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 data sheet suggested a 1: 50 dilution?
  • Fixation
      • Tissue Handling
      • Aldehyde Fixation
      • What is considered the universal reagent used in tissue fixation for IHC staining procedures?
      • Which of the following may occur to the tissue when using 10% NBF fixation for IHC procedures? (Choose all that apply.)
      • Formalin/Aldehyde Substitutes
      • What are some disadvantages to using formalin substitutes? (Choose all that apply.)
      • Additives in alternative fixatives can cause either false negative or false positive staining.
      • Zinc Fixatives
      • How might the fixative action of zinc tris be classified?
      • Which of the following stains may be compromised if zinc tris fixative is used? (Choose all that apply.)
      • Alternative Fixatives for IHC
      • What fixatives/substitutes are NOT the best choice for IHC staining? (Choose all that apply.)
      • What fixative is NOT ideal for IHC staining due to incomplete removal of picric acid?
  • Processing and Microtomy
      • Processing Principles
      • Which of the following is TRUE when handling tissue prior to immuno staining?
      • Frozen Sections
      • For IHC staining, a frozen section is the preferred specimen.
  • Epitope Retrieval
      • General Epitope/Antigen Unmasking
      • Why Perform Epitope/Antigen Unmasking?
      • Which of the following are the two main types of epitope retrieval?
      • Why would it be necessary to unmask antigens?
      • Caused by formalin fixation, what are the strong bonds called that form between proteins and calcium ions?
      • At which point in the 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 an antigen unmasking procedure?
      • Enzyme-Induced Epitope Retrieval (EIER)
      • What type of digestion is enzyme-induced epitope retrieval (EIER)?
      • What two major factors require stringent control when performing enzyme-induced epitope retrieval (EIER) antigen unmasking?
  • Methods of Detection and Visualization
      • Substrates
      • What are substrates? (Choose all that apply.)
      • Chromogens
      • What are the most common chromogens used in IHC? (Choose all that apply.)
      • 3,3’-diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC)
      • Which enzyme substrate is alcohol and solvent resistant?
      • Alkaline Phosphatase
      • When using an IHC alkaline phoshatase staining method, what is the mechanism that produces the color?
      • 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 chromagen enhancer? (Choose all that apply.)
  • Immunohistochemical (IHC) Methods
      • Immunohistochemical (IHC) Techniques
      • Indirect Method
      • What condition is necessary for the secondary antibody in an indirect method?
      • Direct Method
      • The direct method is a multi-step method that uses a labeled antibody that reacts directly with a series of antigen tissue sections.
      • Avidin-Biotin Complex (ABC) Methods
      • The avidin-biotin complex (ABC) technique consists of using which of the following? (Choose all that apply.)
      • Avidin-Biotin Complex (ABC) Method
      • Put the following steps for the ABC method in the correct order for successful staining.
      • Secondary Antibody
      • What is the proper primary-secondary antibody setup when performing an avidin-biotin complex (ABC) procedure?
      • What is the purpose of the secondary antibody? (Choose all that apply.)
      • Which of the following can be used to label avidin?
      • Peroxidase, Anti-Peroxidase (PAP) Method
      • What does PAP stand for?
  • Blocking Methods
      • General Blocking Methods
      • Why are blocking methods used? (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 process are blocking serums utilized?
      • When is a high concentration of blocking serum needed?
      • Blocking Endogenous Peroxidase Activity
      • What does using endogenous peroxidase blocker prevent?
      • What reagents may be used to mix the hydrogen peroxide (H2O2) blocking solution? (Choose all that apply.)
      • Enzyme Blocking
      • 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?
  • Immunohistochemisty (IHC) Staining Reagents
      • General Reagents
      • 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.)
      • What is the proper way to handle 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?
      • 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 as far as use of negative control?
      • Positive Quality Control (QC) Slides
      • Why is it necessary to use a known positive control?
      • System Quality Control (QC)
      • Which antibody is MOST commonly used as a system control?
      • Antibody Validations
      • What are some important factors to consider when validating 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
      • 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: 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.
 
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. 
 
Reviewer information:  Tanisha N. Neely, HT(ASCP) is a freelance writer with a work history in histology, microbiology, and pharmaceutical research. Ms. Neely attended Indiana University where she earned a bachelors degree in arts & humanities from the School of General Studies and a Certificate in Histotechnology from the School of Medicine. She is a member of the National Society for Histotechnology and the Indiana Society for Histotechnology.
 
Course descripiton:  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.

Immunohistochemistry (IHC) Basics in Histology Keywords

These are the most common topics and keywords covered in Immunohistochemistry (IHC) Basics in Histology:

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Alcohol fixed with background


well defined IHC


IHC Counterstains


Image provide by Jim Burchette, HT(ASCP)