An Optimized Protocol for Multiple Immunohistochemical Staining of Fragile Tissue Samples

Area of Science:

• Biomedical Engineering
• Histology
• Immunohistochemistry

Background:

• Multiplex immunohistochemistry (mIHC) is crucial for analyzing multiple biomarkers simultaneously.
• Fragile tissues, including tendons, ligaments, and bones, are prone to detachment during sample preparation, limiting mIHC applications.
• Optimizing tissue section preparation is essential for reliable mIHC results on delicate samples.

Purpose of the Study:

• To develop and optimize a method for preparing fragile tissue sections for multiplex immunohistochemistry (mIHC).
• To evaluate the effectiveness of ultraviolet (UV) light-induced cross-linking compared to traditional baking methods for preserving tissue integrity.
• To determine the optimal UV cross-linking duration for maintaining tissue morphology during mIHC.

Main Methods:

• Paraffin-embedded human anterior cruciate ligament sections were divided into baking and UV cross-linking groups.
• Baking groups were subjected to 65°C for 4, 8, or 24 hours.
• UV cross-linking groups used adhesive slides and were exposed to UV light for 0s to 5 minutes, followed by simulated microwave steps and eosin staining.

Main Results:

• UV cross-linking for 1-4 minutes, unlike baking or shorter/longer UV exposure, maintained complete tissue morphology and structure after simulated mIHC processing.
• The 0s UV group showed complete detachment, while baking groups exhibited significant detachment after microwave treatment.
• Spectral imaging confirmed intact tissue morphology and structure with 2 minutes of UV cross-linking.

Conclusions:

• UV light-induced photocrosslinking is a superior method for preserving fragile tissue integrity during mIHC sample preparation.
• An optimal UV cross-linking time of 1-4 minutes effectively prevents tissue detachment in delicate samples like tendons.
• This optimized method enhances the applicability of mIHC for studying fragile tissues.