A number of conditions can result in an Alcian blue stain for mucin showing excessive background staining.
Causes of excessive background staining:
(1) pH of the staining solution was greater than 2.5
(2) there were impurities in the stain (salt or dextrin)
(3) the time in the stain was too long
(4) the staining solution was too concentrated (perhaps due to a different lot or manufacturer)
(5) failure to adequately wash in 3% acetic acid after the staining step
Steps to ensure proper background staining:
(1) pH of the solution should be less than 2.5
(2) the concentration of the stain should not be too concentrated
(3) high quality stains should be used
(4) contaminated staining solutions should be discarded
(5) the time in the stain should be optimized
(6) rinse properly in 3% acetic acid after the staining step
