Abstract

Immunohistochemistry in fresh-frozen post-fixed brain tissue using antigen retrieval techniques

Krolewski DM, Ilagan RA, Kumar V, Parsegian A, Martin B, Akil H, Watson SJ
Society for Neuroscience. 2016.

Background: Cataloging fresh-frozen brain tissue in the absence of prior aldehyde fixation and cryoprotection is beneficial for preserving protein and nucleic acids (RNA/DNA) across a variety of neuroscience methods requiring long-term storage. Unfortunately, detecting brain-localized antigens via immunohistochemistry (IHC) can be more challenging, if not impossible, using post-fixation following cryosectioning and slide-mounting. The inherent effect of autolysis, particularly during the thawing process, likely contributes to such marked loss of antigenicity (Jones et al., 1992). Slowly thawing frozen human brain blocks immersed in at subfreezing aldehyde-based fixative containing cryoprotectants can retain some degree of antigenicity otherwise lost under less ideal conditions (Jones et al., 1992). However, this technique also can require long incubation times in fixative/cyroprotectant for which thorough tissue penetration can be difficult to determine.
Methods and Results: The present study evaluates more timely IHC methods in an effort to increase antigenicity using fresh-frozen rodent and human brain tissue. The effects of aldehyde-based and non-aldehyde precipitating fixatives were compared across an array of antibodies directed at methylation markers, glial cells, neuropeptides, and other neuronal-specific molecules on slide-mounted sections. In addition, each fixative was tested alone or paired with treatment of heated antigen-retrieval buffers at varied pH. Utilizing standard diaminobenzidine/horseradish peroxidase IHC amplification methods, we show that several antibodies including those specific for glia and particular subcortical neuronal populations are clearly expressed in well-characterized topographical patterns with relatively low background. Given the successful application in thin slices, we have begun conducting similar experiments in 1 to 2mm-thick rodent and human brain slices using transparency techniques. 3-dimensional immunofluorescence in frozen tissue processed by CLARITY (Tomer et al., 2015), CUBIC (Susaki et. al., 2014), and SWITCH methods (Murray et. al., 2015) is being performed using both confocal and CLARITY Optimized Light Sheet Microscopy (COLM) systems. The results of this study will offer additional avenues for the processing of long-term stored fresh-frozen tissue.

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