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A. Peral, J. Pintor; Visualization of Mucins From Human Conjunctiva Combining Impression Cytology and Laser Scanning Microscopy . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1343.
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To visualise the mucins released by goblet cells combing impression cytology and laser scanning microscopy techniques.
Healthy individuals were chosen for this study. Impression cytology was carried out after administration of a topical anaesthesia with proparacaine. Strips of cellulose acetate filter paper (Millipore HAWP304) of 5x5mm were then applied on the conjunctiva for 3 seconds. The filter material was fixed in 96% ethanol, stained with PAS–hematoxylin, dehydrated in ascending grades of ethanol, then xylene and permanently mounted. For the visualization of the impression cytology samples a Zeiss LSM Pascal microscope was used. Magnifications of 20X and 40X (oil) and 60X (oil) were used for the whole set of experiments. Laser scanning microscopy was performed by exciting the samples with the 488 nm and 546 nm wavelength lasers to make PAS–hematoxilyn produce red and green fluorescence respectively. Staking images were taken in order to generate 3–D images suitable to measurements and quantifications.
Light microscopy of impression cytology samples permits to observe conjunctival epithelial cells (pink) and goblet cells (reddish purple), clearly differentiated in terms of shape and colour. The excitation of both pigments with the laser lines permitted us to identify epithelial cells in green and goblet cells in red. Moreover, when performing a stacking followed by a 3–D reconstruction it was possible to see the mucins surrounding goblet cells forming a cloud above them. After this reconstruction in was also possible to measure the main characteristics of these clouds being these values: 2,77±0,20µm for the spreaded mucine over the cell layer and 6,81±0,80µm for the mucine cloud height.
This new approach to visualize mucins may help to estimate the mucin cloud volumes and to better understand the ocular surface pathologies where the amount or the quality of these proteins could be altered.
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