MIBI staining v2

This protocol is the standard FFPE tissue staining procedure recommended for Multiplex Ion Beam Imaging Time of Flight instrument (MIBI_TOF) developed in the Sean C. Bendall and Michael R. Angelo labs. The protocol has been successfully used for MIBI and is the result of extensive optimization experiments. It is inspired from state-of-the art of immunohistochemistry staining procedures but differs in some very important steps, namely, glutaraldehyde fixation and final washes prior tissue dehydration. Failure to follow exactly all steps described in this procedure may result in inconsistencies in output data after MIBI_TOF acquisition.

Sterilization and Cross-Linking Combined with Ultraviolet Irradiation and Low-Energy Electron Irradiation Procedure: New Perspectives for Bovine Pericardial Implants in Cardiac Surgery

Background Bovine pericardium is the major natural source of patches and aortic valve substitutes in cardiac repair procedures. However, long-term tissue durability and biocompatibility issues lead to degeneration (e.g., calcification) that requires reoperation. Tissue preparation strategies, including glutaraldehyde fixation, are reasons for the deterioration of pericardial tissues. We describe a pretreatment procedure involving sterilization and cross-linking combined with ultraviolet (UV) irradiation and low-energy electron irradiation (SULEEI). This innovative, glutaraldehyde-free protocol improves the mechanical aspects and biocompatibility of porcine pericardium patches. Methods We adopted the SULEEI protocol, which combines decellularization, sterilization, and cross-linking, along with UV irradiation and low-energy electron irradiation, to pretreat bovine pericardium. Biomechanics, such as ultimate tensile strength and elasticity, were investigated by comparing SULEEI-treated tissue with glutaraldehyde-fixed analogues, clinical patch materials, and an aortic valve substitute. Histomorphological and cellular aspects were investigated by histology, DNA content analysis, and degradability. Results Mechanical parameters, including ultimate tensile strength, elasticity (Young's modulus), and suture retention strength, were similar for SULEEI-treated and clinically applied bovine pericardium. The SULEEI-treated tissues showed well-preserved histoarchitecture that resembled all pericardial tissues investigated. Fiber density did not differ significantly. DNA content after the SULEEI procedure was reduced to less than 10% of the original tissue material, and more than 50% of the SULEEI-treated pericardium was digested by collagenase. Conclusion The SULEEI procedure represents a new treatment protocol for the preparation of patches and aortic valve prostheses from bovine pericardial tissue. The avoidance of glutaraldehyde fixation may lessen the tissue degeneration processes in cardiac repair patches and valve prostheses.

Glutaraldehyde: A review of its fixative effects on nucleic acids, proteins, lipids, and carbohydrates

Tissue banking methods such as brain banking often face a trade-off between morphological and molecular preservation. For example, cryopreservation is preferred for subsequent molecular assays, while fixation by aldehyde crosslinking is commonly used for microscopy. Among aldehyde fixatives, formaldehyde is often considered better for immunohistochemistry, while glutaraldehyde is often considered better for electron microscopy. However, it is unclear whether morphological versus molecular preservation trade-offs reflect fundamental biology or technology limitations. As a window into this discussion, in this narrative review, I evaluate the literature regarding the effects of glutaraldehyde on molecular preservation, with an emphasis on nervous system tissue. Available evidence suggests that crosslinking with glutaraldehyde has minimal direct effects on most molecular features, with a few critical exceptions such as protein conformation. On the other hand, glutaraldehyde fixation frequently fails to retain non-directly crosslinked molecules such as lipid and carbohydrate species during subsequent dehydration steps. Further, as a result of probe diffusion limitations or strong covalent interactions with glutaraldehyde, many molecules can be more difficult to visualize or otherwise measure in tissue fixed with glutaraldehyde. As a practical guide for investigators that are considering using glutaraldehyde, I also point out representative molecular assays that have been performed in tissue fixed with glutaraldehyde, and how this set of assays could be improved and expanded in the future.