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Published on: April 3, 2013
Immunolabeling recovery in archival, post-mortem, human brain tissue using modified antigen retrieval and the
Joan Y W Liu1, Lillian Martinian, Maria Thom
1Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology and National Hospital for Neurology and Neurosurgery, 33 Queen Square, London WC1N 3BG, UK. j.liu@ion.ucl.ac.uk
Insights
A new protocol using citrate and formic acid pre-treatments with catalyzed signal amplification (CSA) can restore lost immunolabeling in long-formalin-fixed human brain tissue. This method overcomes formalin masking, enabling research with valuable, aged tissue samples.
Area of Science:
- Neuroscience
- Immunohistochemistry
- Biochemistry
Background:
- Human brain tissue is crucial for research but formalin fixation can degrade antibody binding.
- Long-term storage of formalin-fixed tissue (up to 10 years) at room temperature often weakens immunolabeling.
- Formalin-induced masking of antigens poses a challenge for immunohistochemical studies.
Purpose of the Study:
- To develop and validate a novel protocol to recover lost or weakened immunolabeling in long-term formalin-fixed human brain tissue.
- To assess the efficacy of the protocol for various formalin-sensitive antibodies.
- To enable accurate interpretation of immunohistochemical results using aged human brain samples.
Main Methods:
- A novel protocol combining citrate and formic acid pre-treatments was developed.
- The catalyzed signal amplification (CSA) system was integrated into the protocol.
- The protocol was tested on post-mortem human brain tissue fixed in formalin for up to 10 years at room temperature.
- Immunolabeling was assessed for formalin-sensitive antibodies including anti-CD34, anti-caveolin, anti-P-glycoprotein, anti-neuronal nuclei, anti-parvalbumin, anti-human leukocyte antigen, anti-CD45, anti-CD68, and anti-connexin 43.
Main Results:
- The novel protocol successfully recovered lost or weakened immunolabeling for all tested formalin-sensitive antibodies.
- Recovered immunolabeling in long-fixed tissue closely resembled that observed in tissue fixed for shorter durations (6-49 days).
- The method effectively overcame formalin-masking of antigens in aged human brain tissue.
Conclusions:
- The developed protocol is effective in restoring immunolabeling in long-term formalin-fixed human brain tissue.
- Testing antibodies for formalin fixation effects is crucial, especially when using aged tissue.
- This method provides a valuable tool for conducting essential immunohistochemical studies on precious, long-fixed human brain samples.
Abstract:
Human brain tissue is a valuable source of material for research. It is often stored indefinitely in formalin at room temperature which may weaken the immunolabeling with formalin-sensitive antibodies. The present study found that a novel protocol that combined citrate and formic acid pre-treatments with the catalyzed signal amplification (CSA) system was able to recover the lost or weakened immunolabeling with the formalin-sensitive antibodies, anti-CD34, anti-caveolin, anti-P-glycoprotein, anti-neuronal nuclei, anti-parvalbumin, anti-human leukocyte antigen, anti-CD45, anti-CD68 and anti-connexin 43, in post-mortem, human brain tissue that was stored in formalin for up to 10 years at room temperature. Recovered immunolabeling in long-fixed tissue resembled immunolabeling observed in tissue that was fixed for a shorter duration between 6 and 49 days. The findings from this study highlight the importance of testing antibodies for formalin fixation effect prior to studies, especially if long-fixed tissue is used, to enable immunolabeling to be more accurately interpreted. Importantly, this study provides a method of overcoming formalin-masking of antigens in long-fixed human tissue, thus allowing essential immunohistochemical studies to be undertaken using precious human tissue.
