I manage the advanced microimaging facility at the Queensland Brain Institute, University of Queensland. Some of the imaging undertaken here includes large scale imaging of brain tissue and high-resolution imaging of neurons and cellular dynamics.
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Nickel DAB (N-DAB) Labeling for sections
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Neuron recovery of neurobiotin/biocytin filled neurons. The method may applied to free-floating or cryostat sectioned fixed tissue.
This technique results in a black insoluble reaction product delineating the filled neuron. Developing the chromagen is slow and should be monitored microscopically. The reaction is stopped before the background staining occurs. The labeled sections can then be counter stained and mounted permanently.
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Haemotoxylin and eosin stain is used to stain cell nuclei blue and the cytoplasm pink/red to aid visualization of tissue structure and morphology.
The staining method involves application of the basic dye haematoxylin, which stains basophilic structures, usually the ones containing nucleic acids, such as the ribosomes and the chromatin and alcohol-based acidic eosin Y, which colors eosinophilic structures, generally composed of intracellular or extracellular protein, bright pink to yellow.
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8% Paraformaldehyde for Immunofluorescence
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Chrome Alum Gelatin Slide Coating
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10mM Sodium Citrate, 0.05% Tween 20, pH 6.0
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Aqueous mounting medium for fluorescence and Stereology. Recommended Anti-Fade for fluorescence microscopy – especially good for EGFP and other low expression reporting molecules.
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Dewaxing Paraffin Sections Sections must be free of wax to allow aqueous solutions to penetrate.
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Luxol Fast Blue histological stain. This technique is a method requiring over-staining of tissues and differentiation to reveal the myelin. It is advisable to include a control showing de-myelination pathology to ensure the end point of staining is reached. Cresyl Fast Violet counter-stain is optional and results in neuronal piliform staining and a deepening of the myelin blue staining.
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Cresyl Violet Staining for free floating sections, mounted and air-dried. Cresyl Violet Acetate solution is used to stain Nissl substance in the cytoplasm of neurons in paraformaldehyde or formalin-fixed tissue. The neuropil will be stained a granular purple-blue. This stain is commonly used to identify the neuronal structure in brain and spinal cord tissue.
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