Langkah Prosedur Pewarnaan Imunohistokimia (IHK)

Introduction

This tutorial provides a comprehensive guide to the immunohistochemical staining procedure as demonstrated in the video by Prof. Dr. Drs. Kusmardi. Immunohistochemistry (IHC) is a crucial technique in both research and clinical settings for detecting specific antigens in tissues. Understanding this procedure can enhance your skills in histology and pathology.

Step 1: Prepare Tissue Samples

• Collect and fix tissue samples using an appropriate fixative (e.g., formalin).
• Embed the samples in paraffin wax to create a solid block that can be sliced into thin sections.
• Cut sections of 3-5 micrometers using a microtome and place them onto glass slides.

Step 2: Deparaffinization and Rehydration

• Heat the slides to melt the paraffin, typically at around 60°C for 30 minutes.
• Immerse the slides in xylene for 2-3 minutes to remove the paraffin.
• Rehydrate the sections by passing them through a series of ethanol solutions:
  • 100% ethanol (2 minutes)
  • 95% ethanol (2 minutes)
  • 70% ethanol (2 minutes)
  • Rinse with distilled water.

Step 3: Antigen Retrieval

• Perform antigen retrieval to unmask antigens that may have been masked during fixation. This can be done using:
  • Heat-induced epitope retrieval (HIER) with a citrate buffer solution.
  • Enzymatic retrieval using proteolytic enzymes like trypsin.
• Incubate the slides in the retrieval solution, then cool them at room temperature.

Step 4: Blocking Non-Specific Binding

• Incubate the tissue sections with a blocking solution (e.g., serum or BSA) to prevent non-specific binding of antibodies.
• Allow the blocking solution to sit for 30 minutes to 1 hour at room temperature.

Step 5: Primary Antibody Incubation

• Dilute the primary antibody in an appropriate buffer.
• Apply the diluted primary antibody to the tissue sections and incubate for 1-2 hours at room temperature or overnight at 4°C.
• Wash the slides with phosphate-buffered saline (PBS) or Tris-buffered saline (TBS) to remove unbound antibodies.

Step 6: Secondary Antibody Application

• Apply a secondary antibody that is conjugated to a detection enzyme (e.g., horseradish peroxidase).
• Incubate for 30-60 minutes at room temperature.
• Wash the slides again with PBS or TBS.

Step 7: Visualization of Staining

• Prepare a substrate solution for the detection enzyme (e.g., DAB for HRP).
• Apply the substrate to the slides and incubate until the desired color intensity is reached.
• Stop the reaction by rinsing with distilled water.

Step 8: Counterstaining

• Use a counterstain (e.g., hematoxylin) to provide contrast to the primary staining.
• Apply the counterstain for a brief period, then rinse with water.

Step 9: Mounting

• Dehydrate the slides by passing them through increasing concentrations of ethanol.
• Clear the slides with xylene and apply a mounting medium.
• Cover with a coverslip and allow it to set.

Conclusion

The immunohistochemical staining procedure involves several critical steps, from sample preparation to visualization. Mastering this technique can significantly enhance your understanding and analysis of tissue samples in both research and clinical settings. For further practice, consider applying this procedure with different antibodies or exploring variations in staining protocols.