Mustafa S Kassem1, Sandra Y Y Fok2, Kristie L Smith3, Michael Kuligowski4, Bernard W Balleine5. 1. Decision Neuroscience Lab, School of Psychology, University of NSW, NSW, 2052 Australia; Brain and Mind Research Centre, University of Sydney, NSW, 2006, Australia. 2. Biomedical Imaging Facility, University of NSW, 2052, Australia. 3. Brain and Mind Research Centre, University of Sydney, NSW, 2006, Australia. 4. Australian Microscopy & Microanalysis Research Facility at the Australian Centre for Microscopy & Microanalysis, University of Sydney, NSW, 2006 Australia. 5. Decision Neuroscience Lab, School of Psychology, University of NSW, NSW, 2052 Australia. Electronic address: bernard.balleine@unsw.edu.au.
Abstract
BACKGROUND: High resolution neuronal information is extraordinarily useful in understanding the brain's functionality. The development of the Golgi-Cox stain allowed observation of the neuron in its entirety with unrivalled detail. Tissue clearing techniques, e.g., CLARITY and CUBIC, provide the potential to observe entire neuronal circuits intact within tissue and without previous restrictions with regard to section thickness. NEW METHOD: Here we describe an improved Golgi-Cox stain method, optimised for use with CLARITY and CUBIC that can be used in both fresh and fixed tissue. RESULTS: Using this method, we were able to observe neurons in their entirety within a fraction of the time traditionally taken to clear tissue (48h). We were also able to show for the first-time that Golgi stained tissue is fluorescent when visualized using a multi-photon microscope, allowing us to image synaptic spines with a detail previously unachievable. COMPARISON WITH EXISTING METHODS: These novel methods provide cheap and easy to use techniques to investigate the morphology of cellular processes in the brain at a new-found depth, speed, utility and detail, without previous restrictions of time, tissue type and section thickness. CONCLUSIONS: This is the first application of a Golgi-Cox stain to cleared brain tissue, it is investigated and discussed in detail, describing different methodologies that may be used, a comparison between the different clearing techniques and lastly the novel interaction of these techniques with this ultra-rapid stain.
BACKGROUND: High resolution neuronal information is extraordinarily useful in understanding the brain's functionality. The development of the Golgi-Cox stain allowed observation of the neuron in its entirety with unrivalled detail. Tissue clearing techniques, e.g., CLARITY and CUBIC, provide the potential to observe entire neuronal circuits intact within tissue and without previous restrictions with regard to section thickness. NEW METHOD: Here we describe an improved Golgi-Cox stain method, optimised for use with CLARITY and CUBIC that can be used in both fresh and fixed tissue. RESULTS: Using this method, we were able to observe neurons in their entirety within a fraction of the time traditionally taken to clear tissue (48h). We were also able to show for the first-time that Golgi stained tissue is fluorescent when visualized using a multi-photon microscope, allowing us to image synaptic spines with a detail previously unachievable. COMPARISON WITH EXISTING METHODS: These novel methods provide cheap and easy to use techniques to investigate the morphology of cellular processes in the brain at a new-found depth, speed, utility and detail, without previous restrictions of time, tissue type and section thickness. CONCLUSIONS: This is the first application of a Golgi-Cox stain to cleared brain tissue, it is investigated and discussed in detail, describing different methodologies that may be used, a comparison between the different clearing techniques and lastly the novel interaction of these techniques with this ultra-rapid stain.