Alexander J Ehrenberg1, Dulce Ovando Morales2, Antonia M H Piergies2, Song Hua Li2, Jorge Santos Tejedor3, Mihovil Mladinov2, Jan Mulder4, Lea T Grinberg5. 1. University of California, San Francisco, Memory and Aging Center, Weill Institute for Neurosciences; San Francisco, CA, USA; University of California, Berkeley, Helen Wills Neuroscience Institute; Berkeley, CA, USA; University of California, Berkeley, Dept. of Integrative Biology; Berkeley, CA, USA. 2. University of California, San Francisco, Memory and Aging Center, Weill Institute for Neurosciences; San Francisco, CA, USA. 3. University of California, San Francisco, Memory and Aging Center, Weill Institute for Neurosciences; San Francisco, CA, USA; Karolinska Instituet, Department of Neuroscience, Stockholm, Sweden. 4. Karolinska Instituet, Department of Neuroscience, Stockholm, Sweden. 5. University of California, San Francisco, Memory and Aging Center, Weill Institute for Neurosciences; San Francisco, CA, USA; University of São Paulo School of Medicine, São Paulo, Brazil; University of California, San Francisco, Global Brain Health Institute; San Francisco, CA, USA. Electronic address: lea.grinberg@ucsf.edu.
Abstract
BACKGROUND: Neurodegenerative diseases feature stereotypical deposits of protein aggregates that selectively accumulate in vulnerable cells. The ability to simultaneously localize multiple targets in situ is critical to facilitate discovery and validation of pathogenic molecular pathways. Immunostaining methods enable in situ detection of specific targets. Effective stripping of antibodies, allowing successive rounds of staining while maintaining tissue adhesion and antigen integrity, is the main roadblock for enabling multiplex immunostaining in standard labs. Furthermore, stripping techniques require antibody-specific optimization, validation, and quality control steps. NEW METHOD: Aiming to create protocols for multiplex localization of neurodegenerative-related processes, without the need for specialized equipment, we evaluated several antibody stripping techniques. We also recommend quality control steps to validate stripping efficacy and ameliorate concerns of cross-reactivity and false positives based on extensive testing. RESULTS: A protocol using β-mercaptoethanol and SDS consistently enables reliable antibody stripping across multiple rounds of staining and minimizes the odds of cross-reactivity while preserving tissue adhesion and antigen integrity in human postmortem tissue. COMPARISON WITH EXISTING METHODS: Our proposed method is optimal for standard lab settings and shows consistent efficacy despite the intricacies of suboptimal human postmortem tissue and the need to strip markers bound to highly aggregated proteins. Additionally, it incorporates quality control steps to validate antibody stripping. CONCLUSIONS: Multiplex immunofluorescence methods for studying neurodegenerative diseases in human postmortem tissue are feasible even in standard laboratories. Nevertheless, evaluation of stripping parameters during optimization and validation phases of experiments is prudent.
BACKGROUND:Neurodegenerative diseases feature stereotypical deposits of protein aggregates that selectively accumulate in vulnerable cells. The ability to simultaneously localize multiple targets in situ is critical to facilitate discovery and validation of pathogenic molecular pathways. Immunostaining methods enable in situ detection of specific targets. Effective stripping of antibodies, allowing successive rounds of staining while maintaining tissue adhesion and antigen integrity, is the main roadblock for enabling multiplex immunostaining in standard labs. Furthermore, stripping techniques require antibody-specific optimization, validation, and quality control steps. NEW METHOD: Aiming to create protocols for multiplex localization of neurodegenerative-related processes, without the need for specialized equipment, we evaluated several antibody stripping techniques. We also recommend quality control steps to validate stripping efficacy and ameliorate concerns of cross-reactivity and false positives based on extensive testing. RESULTS: A protocol using β-mercaptoethanol and SDS consistently enables reliable antibody stripping across multiple rounds of staining and minimizes the odds of cross-reactivity while preserving tissue adhesion and antigen integrity in human postmortem tissue. COMPARISON WITH EXISTING METHODS: Our proposed method is optimal for standard lab settings and shows consistent efficacy despite the intricacies of suboptimal human postmortem tissue and the need to strip markers bound to highly aggregated proteins. Additionally, it incorporates quality control steps to validate antibody stripping. CONCLUSIONS: Multiplex immunofluorescence methods for studying neurodegenerative diseases in human postmortem tissue are feasible even in standard laboratories. Nevertheless, evaluation of stripping parameters during optimization and validation phases of experiments is prudent.
Authors: Panos Theofilas; Antonia M H Piergies; Ian Oh; Yoo Bin Lee; Song Hua Li; Felipe L Pereira; Cathrine Petersen; Alexander J Ehrenberg; Rana A Eser; Andrew J Ambrose; Brian Chin; Teddy Yang; Shireen Khan; Raymond Ng; Salvatore Spina; Willian W Seeley; Bruce L Miller; Michelle R Arkin; Lea T Grinberg Journal: Neuropathol Appl Neurobiol Date: 2022-06-02 Impact factor: 6.250