The two methods for measuring mucin production in vivo that will be described below utilize microscopic imaging and biochemical assays.
Histological specimens provide a useful tool to determine the localization and degrees of mucin production and secretion. For measuring intracellular mucin content, a technique such as transmission electron microscopy is exquisitely sensitive. However, instrument expense and sample preparation time can be prohibitive when large numbers of samples are assessed. For these reasons, we have relied more heavily on light microscopy and the use of inexpensive technologies to measure mucin production and secretion. Images obtained at relatively low magnification (e.g. using a 40 x specimen objective), can be analyzed and compared at different anatomical locations in the same slide, provide adequate sample sizes (10’s to 100’s of cells per image), and display sufficient detail to determine whether there is heterogeneity among cells. Numerous image analysis software tools make quantitation of staining simple and inexpensive.
Immunoblotting is also an efficacious approach for measuring the airway mucin content. Due to particular biochemical properties of polymeric mucins, treatment with a chaotropic agent such as guanidinium chloride is necessary for breaking non-covalent bonds and solubilization (20 (link)). Polymeric mucins are also held together by disulfide bonds. Therefore, it is important to reduce these with agents such as dithiothreitol prior to electrophoresis and transfer (21 (link)). These procedures will permit resolution of single bands of monomeric mucins. Mucins can then be blotted and detected using selective probes. Their heavy glycosylation has made use of specific antibodies difficult in some instances (22 ), but this same property makes mucins suitable for detection with lectins, a class of highly specific sugar binding proteins. Results can be compared relative to each other within blots or across different vacuum blots when a standard curve and appropriate internal controls are also applied.
Histological specimens provide a useful tool to determine the localization and degrees of mucin production and secretion. For measuring intracellular mucin content, a technique such as transmission electron microscopy is exquisitely sensitive. However, instrument expense and sample preparation time can be prohibitive when large numbers of samples are assessed. For these reasons, we have relied more heavily on light microscopy and the use of inexpensive technologies to measure mucin production and secretion. Images obtained at relatively low magnification (e.g. using a 40 x specimen objective), can be analyzed and compared at different anatomical locations in the same slide, provide adequate sample sizes (10’s to 100’s of cells per image), and display sufficient detail to determine whether there is heterogeneity among cells. Numerous image analysis software tools make quantitation of staining simple and inexpensive.
Immunoblotting is also an efficacious approach for measuring the airway mucin content. Due to particular biochemical properties of polymeric mucins, treatment with a chaotropic agent such as guanidinium chloride is necessary for breaking non-covalent bonds and solubilization (20 (link)). Polymeric mucins are also held together by disulfide bonds. Therefore, it is important to reduce these with agents such as dithiothreitol prior to electrophoresis and transfer (21 (link)). These procedures will permit resolution of single bands of monomeric mucins. Mucins can then be blotted and detected using selective probes. Their heavy glycosylation has made use of specific antibodies difficult in some instances (22 ), but this same property makes mucins suitable for detection with lectins, a class of highly specific sugar binding proteins. Results can be compared relative to each other within blots or across different vacuum blots when a standard curve and appropriate internal controls are also applied.
