Immunohistochemistry (IHC) Protocol (Paraffin-Embedded Tissue)
IImmunohistochemistry (IHC) is a powerful technique widely used in research and clinical laboratories to detect and visualize specific proteins or antigens within preserved tissue sections.
This protocol is specifically designed for paraffin-embedded tissue samples, a common preparation method that allows for long-term storage and detailed morphological analysis.The IHC protocol involves a series of carefully orchestrated steps, including tissue fixation, paraffin embedding, sectioning, deparaffinization, rehydration, antigen retrieval, and specific antibody staining. Each step is crucial in ensuring the preservation of tissue architecture, the accessibility of target antigens, and the specificity and sensitivity of antibody binding.
This comprehensive guide provides a standardized protocol that consolidates best practices from various methodologies to ensure reproducible and high-quality results. By following this protocol, researchers can achieve reliable staining patterns that highlight the presence and localization of specific proteins within tissue samples, aiding in the study of cellular processes, disease pathogenesis, and biomarker discovery.
Whether you are performing basic research or diagnostic investigations, this IHC protocol for paraffin-embedded tissues provides a robust framework for successful immunohistochemical staining and analysis.
Immunohistochemistry (IHC) Protocol (Paraffin-Embedded Tissue)
Steps 1-8: Preparing Formalin-Fixed, Paraffin-Embedded Tissue Sections
1. Fixation.Fix freshly dissected tissue (<3 mm thick) with 2-4% paraformaldehyde for 1 hour to overnight at room temperature.
2. Rinsing: Rinse the tissue with running tap water for 5 minutes..
3. Dehydration. Dehydrate the tissue through graded alcohols: 70%, 80%, and 95% ethanol for 5 minutes each, followed by 100% ethanol (three times, 5 minutes each).
4. Clearing:Clear the tissue in xylene (two times, 5 minutes each).
5. Paraffin Immersion: Immerse the tissue in hot paraffin (three times, 5 minutes each) at 50–60°C.
6. Embedding: Embed the tissue in a paraffin block. The paraffin-embedded tissue block can be stored at room temperature for years.
7. Sectioning: Section the paraffin-embedded tissue block at a thickness of 5-8 µm using a microtome and float sections in a 40°C water bath containing distilled water.
8. Mounting: Transfer sections onto glass slides suitable for immunohistochemistry. Allow slides to dry overnight and store at room temperature until ready for use.
Steps 9-29: Immunostaining Formalin-Fixed, Paraffin-Embedded Tissue Sections
9. Deparaffinization: Deparaffinize slides in xylene (two times, 5 minutes each).
10. Rehydration: Rehydrate slides through 100% ethanol (two times, 3 minutes each), followed by 95%, 70%, and 50% ethanol (3 minutes each).
11. Blocking Endogenous Peroxidase:Incubate sections in 3% H2O2 solution in methanol at room temperature for 10 minutes to block endogenous peroxidase activity.
12. Rinsing:Rinse with PBS (two times, 5 minutes each).
13. Antigen Retrieval (Optional, Recommended):Perform antigen retrieval using a citrate buffer method: Place slides in a staining container with 300 ml of 10 mM citrate buffer (pH 6.0). Incubate at 95-100°C for 10 minutes (optimal incubation time should be determined by the user). Allow slides to cool at room temperature for 20 minutes.
14. Rinsing: Rinse slides with PBS (two times, 5 minutes each).
15. Blocking (Optional):Add 100 µl of blocking buffer (e.g., 10% fetal bovine serum in PBS) to sections and incubate in a humidified chamber at room temperature for 1 hour.
16. Draining: Drain off the blocking buffer from the slides.
17. Primary Antibody Incubation: Apply 100 µl of appropriately diluted primary antibody (in antibody dilution buffer, e.g., 0.5% bovine serum albumin in PBS) to sections and incubate in a humidified chamber at room temperature for 1 hour.
18. Washing: Wash slides with PBS (two times, 5 minutes each).
19. Secondary Antibody Incubation: Apply 100 µl of appropriately diluted biotinylated secondary antibody (in antibody dilution buffer) to sections and incubate in a humidified chamber at room temperature for 30 minutes.
20. Washing: Wash slides with PBS (two times, 5 minutes each).
21. Conjugate Incubation: Apply 100 µl of appropriately diluted Streptavidin-HRP conjugate (in antibody dilution buffer) to sections and incubate in a humidified chamber at room temperature for 30 minutes (keep protected from light).
22. Washing: Wash slides with PBS (two times, 5 minutes each).
23. DAB Staining: Apply 100 µl of DAB substrate solution (freshly prepared: 0.05% DAB - 0.015% H2O2 in PBS) to sections to reveal the color of antibody staining. Develop color for less than 5 minutes until the desired intensity is reached. Caution: DAB is a suspected carcinogen; handle with care using gloves, a lab coat, and eye protection.
24. Washing: Wash slides with PBS (three times, 2 minutes each).
25. Counterstaining (Optional):Counterstain slides by immersing them in Hematoxylin for 1-2 minutes.
26. Rinsing: Rinse slides in running tap water for 10 minutes.Dehydration:Dehydrate tissue slides through a series of alcohols (95%, 95%, 100%, 100%), 5 minutes each.Clearing and Mounting:Clear tissue slides in xylene (three times) and coverslip using a mounting solution. Mounted slides can be stored at room temperature indefinitely.
27. Observation: Observe the color of antibody staining in tissue sections under a microscope.
Algent IHC antibodies
Immunohistochemistry (IHC) is an advanced technique in histopathology that plays a crucial role in molecular pathology for diagnosis, prognosis, and prediction. IHC allows for the visualization of the distribution and localization of specific cellular components within a cell or tissue, making it a valuable tool in medical research and diagnostics.