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. Finally, using the optimal cross-linking time selected from the UV cross-linking groups, mIHC staining of tendon and bone tissues was performed. After deparaffinization and rehydration, both groups were able to maintain the integrity of the tissue structure, except for the slides from the UV-sensitive cross-linking 0 s group, which showed complete tissue detachment. Following the seventh microwave treatment, the baking groups presented significant tissue detachment. The UV cross-linking groups were affected by the cross-linking time, and severe tissue detachment occurred with cross-linking times of 20 s, 40 s, and 5 min, whereas the tissues cross-linked for 1 min, 2 min, 3 min, and 4 min all maintained complete tissue morphology and structure. Finally, after 2 min of cross-linking, the results of spectral imaging revealed that the tissue morphology and structure were intact. During the process of mIHC staining, photocrosslinking with UV irradiation for 1-4 min effectively preserves the integrity of the tissue morphological structure.