An Optimized Protocol for Multiple Immunohistochemical Staining of Fragile Tissue Samples.

Owing to the high occurrence of tissue detachment during the sample preparation process, the application of multiplex immunohistochemistry (mIHC) technology is limited in the field of fragile tissue samples, such as tendons, ligaments, and bones. To optimize a method for preparing sections for mIHC on fragile tissue samples, taking the human anterior cruciate ligament as an example, paraffin-embedded continuous sections with a thickness of 4 μm were divided into two groups: baking groups underwent routine section processing, and after being mounted on glass slides, they were baked at 65°C for 4 h, 8 h, or 24 h; ultraviolet (UV) photosensitive cross-linking groups used adhesive-coated slides for mounting and were directly subjected to UV light-induced cross-linking, with the cross-linking time set at 0 s, 20 s, 40 s, 1 min, 2 min, 3 min, 4 min, and 5 min, respectively. After deparaffinization and rehydration, we simulated the microwave step, which was most likely to cause tissue detachment during the mIHC experimental procedure, and then, the sections were stained with eosin.