IHC_Fixation_and_Tissue_Preparation_Presentation[1].pptx

Immunohistochemistry in a histopathology laboratory, ideology of fixation of tissues for IHC, and antigen retrieval methods

1. Fixation and Tissue Preparation in IHC and Antigen Retrieval Method s. Group 5 Presentation Presented by: MATTE ELIJAH INNOCENT 2021/MLS/202/PS ARINAITWE ALBERT 2021/MLS/212/PS TUSHEMERIRWE RUTH 2020/MLS/125/PS OFOYRWOTH INNOCENT 2021/MLS/003 1

2. Recap Immunohistochemistry ( IHC ) is a technique that uses antibodies to detect specific proteins in tissue sections. Purpose : To visualize protein localization, expression, and distribution in tissues. Two Main Types of IHC : Formalin-Fixed, Paraffin-Embedded (FFPE) Frozen Tissue Sections (IHC-Fr) 2

3. Purpose of tissue Fixation in IHC The fixation can help to prevent; Elution Degradation Modification Preserves the position of the antigen . Preserves the secondary and tertiary structure to a possible extent. Provides target for antibody molecules. 3

4. Tissue Fixation in IHC Types of Fixatives in IHC Aldehydes: Formaldehyde, Glutaraldehyde— crosslink proteins to preserve tissue structure . Alcohols: Ethanol, Methanol— denature proteins by removing water molecules. Acetone: Often used with frozen tissue to preserve enzyme and antigen functionality. 4

5. Common Fixatives: - Formaldehyde-based ( crosslinking proteins ) NB: Formaldehyde is the preferred fixative and most of the antibodies available are optimized for use with formaldehyde. - Alcohol-based (precipitating proteins ) 5

6. Key Fixation Techniques Formalin-Fixed, Paraffin-Embedded ( FFPE ) Protocol: - Immersion in 10% formalin (4% formaldehyde). - Dehydration in ethanol and embedding in paraffin. Frozen Tissue Protocol: - Snap freezing in isopentane cooled with dry ice. - Fixation with cold methanol or acetone. 6

7. Formalin based sections: Advantages: good preservation of morphology, even after prolonged fixation economical chemical sterilizes tissue specimens in a more reliable way than precipitating fixatives, particularly for viruses. Carbohydrate antigens are well preserved Cross-linking of protein in situ avoids leaching out of proteins that may diffuse in water or alcohol. Many low–molecular-weight antigens (peptides) are well preserved in tissue by formalin. 7

8. Frozen sections Although the use of frozen sections for diagnostic purposes is decreasing, immunohistochemistry on frozen sections remains an important histological tool. Advantage : preserves enzyme and antigen function Disadvantages: Poor morphology Limited prospective studies Storage of material difficult Cutting difficulty over paraffin sections 8

9. Epitope Retrieval in IHC Types of Antigen Retrieval: - Heat-Induced Epitope Retrieval (HIER) : Uses heat to unmask epitopes. - Proteolytic-Induced Epitope Retrieval (PIER): Uses enzymes to digest proteins and expose epitopes. 9

10. HIER Protocol Used with different buffers; Citrate Buffer (10mm Sodium Citrate 0.05% Tween 20) at pH 6.0) Tris-Buffered Saline pH 9.0 EDTA Buffer at pH 8.0 2. Other requirements microwave heat plate steamer pressure cooker/autoclave 10

11. Procedure(Steps) Put citrate buffer into a pressure cooker or heat it without a lid in the microwave for 5 minutes ( Prewarming ) Put the rack into the citrate buffer Put a lid on the cooker and heat the cooker for 10-15 minutes Remove the rack and cool it down at RT for 30 minutes Rinse the rack with tap water for 10 minutes Dip the slides in PBS buffer (at RT) for 10 minutes 11

12. HIER METHODS HIER METHOD ADVANTANGES DISADVANTAGES Water bath (90-98) gentler on the tissue sections because the temperature is set below boiling point antigen retrieval buffer does not evaporate . Antig e n retri e v a l ti mes are i n cr e ased c o mpared t o oth e r methods. Steamer less damaging to tissues than the other heating methods. Steam heating appears to be less efficient than either microwave oven heating or pressure cooking. Pressure cooker Batch variation and production of hot and cold spots in the microwave oven could be overcome (more uniform than the rest). aluminum pressure cookers are susceptible to corrosion from some of the antigen retrieval buffers-use stainless steel). Microwave Reaches the preferred temperature very quickly. Uneven heating and the production of hot spots. 12

13. Advantages of heat pretreatment Some antigens previously thought lost in routinely processed paraffin- embedded sections are now recovered by heat pretreatment. Many a nti g e n s are r e t rieved b y u n ifo r m h e ating times , r e gard l e s s of length of fixation. Pitfalls of heat pretreatment Care should be taken not to allow the sections to dry after heating, as this destroys antigenicity. The boiling of poorly fixed material also damages nuclear detail. Fibrous and fatty tissues tend to detach from the slide. 13

14. PIER Protocol Enzymes u sed: - Trypsin - Proteinase K Others Chymotrypsin Pepsin protease For those antigens that require enzymatic retrieval rather than HIER, the recommended enzyme and digesting conditions will be clearly indicated on the product datasheet. 14

15. Steps: Steps: 1. Incubate tissue slides in the enzyme solution at 37°C for 10-20 minutes. 2. Cool slides before continuing with the staining process. Principle- Digestion breaks down formalin cross-linking and hence the antigenic sites for a number of antibodies are uncovered Under-digestion results in too little staining, because the antigens are not fully exposed. Over-digestion can produce false positive staining, high background levels, and tissue damage . 15

16. When to Use Each Method? HIER: Preferred for most formalin-fixed tissues; first choice for less cross-linked proteins. PIER: Used for heavily cross-linked tissues where HIER is not effective. 16

17. Challenges in Fixation and Antigen Retrieval Over-fixation: May cause excessive cross-linking, masking epitopes. Under-fixation: May lead to autolysis or tissue degradation. Solution: Properly optimizing fixation time and retrieval methods. 17

18. Conclusion Key Takeaways: Successful IHC depends on proper fixation and antigen retrieval techniques to visualize target proteins accurately. Final Note: Optimal fixation and antigen retrieval protocols vary based on tissue type and antigen properties. 18

19. References Theory & practices of histologycal thecnique – bancroft 5th edition. Immunohistochemical Methods, Fourth Edition – Dake . 19

20. THANK YOU 20