Immunohistochemistry (IHC) Basics in Histology (by LabCE)

(based on 182 customer ratings)

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

Course provided by LabCE.

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.

See more courses in: Histology

Included In These Course Packages

Continuing Education Credits

Florida Board of Clinical Laboratory Personnel Credit Hours - Immunohistochemistry/Advanced Histologic Techniques: 2 hour(s)
Course number 20-547723, approved through 9/1/2020


  • 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 182 customer ratings)

Course Outline

  • The Role of Immunohistochemistry (IHC) in the Histology Laboratory
      • Immunohistochemistry (IHC) Uses and Considerations
      • Antibodies, Antigens, and Epitopes
  • Antibodies
      • Monoclonal Antibodies
      • Polyclonal Antibodies
      • Antibody Titering
  • Fixation
      • Tissue Handling
      • Aldehyde Fixation
      • Formalin/Aldehyde Substitutes
      • Zinc Fixatives
      • Alternative Fixatives for IHC
  • Processing and Microtomy
      • Processing Principles
      • Frozen Sections
  • Epitope Retrieval
      • General Epitope/Antigen Unmasking
      • Why Perform Epitope/Antigen Unmasking?
  • Unmasking Methods
      • Heat-Induced Epitope Retrieval (HIER)
      • Enzyme-Induced Epitope Retrieval (EIER)
  • Methods of Detection and Visualization
      • Substrates
      • Chromogens
      • 3,3’-diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC)
      • Alkaline Phosphatase
      • Chromogen Enhancers
  • Immunohistochemical (IHC) Methods
      • Immunohistochemical (IHC) Techniques
      • Indirect Method
      • Direct Method
      • Avidin-Biotin Complex (ABC) Methods
      • Avidin-Biotin Complex (ABC) Method
      • Secondary Antibody
      • Peroxidase, Anti-Peroxidase (PAP) Method
  • Blocking Methods
      • General Blocking Methods
      • Avidin/Biotin Blocking
      • Serum Blocking
      • Blocking Endogenous Peroxidase Activity
      • Enzyme Blocking
  • Immunohistochemisty (IHC) Staining Reagents
      • General Reagents
      • Antibody Diluents
      • Buffers and Rinses
  • Immunohistochemical (IHC) Controls
      • Quality Control (QC)
      • Tissue Controls/Control Slides
      • Negative Quality Control (QC) Slides
      • Positive Quality Control (QC) Slides
      • System Quality Control (QC)
      • Antibody Validations
  • Troubleshooting
      • Troubleshooting
  • Record Keeping
      • Record Keeping
  • 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.


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

toxic technician microtomy formalin unusable isothiocyanate specimens as-tr phosphates solvents preservation phenolic saline rabbit hazardous alcohol phenols peroxidase antigens areantigen label glucose heated y-shaped trimmed immunoglobulins immunofluorescence antibody ffpe aldehydes antigen mouse bonds methanol mounting silver xylene rinselabel bcip hematoxylin cooker hydroxymethyl epitopes antibodies concentration non-immune soluble paraformaldehyde dako aldehyde immune naphthol-as-phosphate conditions crystals sensitivity false-positives drawback calcium fluorophore blue-black digestion multi-step endogenous antiserum considerations naphthol fitc dehydrate trichrome morphology concerns non-staining pre-mixed 3’-diaminobenzidine anti-mouse pockets mammals fluorescein false-positive enhancers trypsin steamer trapped microwave azide non-self glutaraldehyde diazonium methods immunoglobulin wrinkles settings tumors serums pioneer fixatives over-fixation vegetable tris deparaffinization conjugated phosphatase stainers antibody-antigen ß-galactosidase antigenicity hepatocytes amplification 3-amino-9-ethylcarbazole oxidase rigidity metals compounds methodology avidin-biotin proximal pepsin bridges fails solutionmayers osmium laboratory immunohistochemistry enzyme-induced deionized cytoplasm buffers procedures suggestions ultrapure substitutes heterogeneous as-bi monoclonal 3-diaminobenzidine buffer harshest eier biotinylated anti-peroxidase peroxide immunohistochemical waterenzyme labeled enzyme salts cells culture rbcs taylor treatment kidney tubules heat-induced bouins sodium one-step stainingblockprimary artifact mammal controls hydrolyzes methylene diluents solvent catalyzes purer oven potassium chromogen formalin-fixed stirring nuclear fuchsin polyclonal pretreatment unmasking nickel competency peroxidase-based 5-bromo-4-chloro-3-indoxyl gomoris picric serum alcohols validating isotype inconsistency cobalt acetic peroxidase-labeled enzymatic detachment quench non-specific tetroxide reagents epitope paraffin-embedded compliance b-cell horseradish fluorescent non-formalin hydrogen waterantigen cross-links zinc autolysis goats levamisole phosphate enzymes mallorys zenkers coverslipping vimentin dilutions incubation antibodys specificity b-lymphocytes lymph false-negative mount goat injecting penetration labels blocker biotin oligosaccharide #223-galactosidase avidin antibodybuffer fixative hier oxygen samples alkaline resinous chromogens chemical alcohol-based synthetic trap adhesion protein substrate pretreatmentantibody polyclonals executed
How to Subscribe
Histology CE (by LabCE)
Package of 3 online courses$65
ASCLS members save $10
Add to cart
Single online course$25
Add to cart
  • Order for instant access through any computer, any browser.
  • No shipping, faxing, or waiting for certificates.
  • Print or save your certificate of completion as soon as you've completed the course.
  • You'll have 90 days to complete your purchased courses.
Course provided by LabCE.
Image provide by Jim Burchette, HT(ASCP)

Alcohol fixed with background

IHC Counterstains

well defined IHC

ASCLS CE's design and platform are provided by MediaLab, Inc.