Mast cell fixation and staining in image analysis.

Modern image analysers automatically perform densitometric measurements and elaborate digital images. Elaboration however is subject to operator interpretation and often eliminates precious information from the areas of interest. For this reason, it was appropriate to find a staining method which would overcome this drawback and, in the case of mast cell histochemistry, limit staining to granule content. The following current staining techniques were tested: Toluidine Blue in buffered solution (solut. a) and in 0.003% alcoholic solution (solut. b) and alcoholic Astra Blue, pH 0.2 Densitometric analysis was performed on both 5 microns and semithin sections of mouse tongue fixed in Isotonic formaldehyde-acetic acid (IFAA). Digital images were obtained using 630 nm and 546 nm wavelengths for Toluidine Blue and 610 nm for Astra Blue. Direct comparison between the two Toluidine Blue solutions revealed that more pixels were captured by the 5 microns sections stained with solut. a, whilst the opposite occurred in semithin sections. Both dyes introduced a certain amount of error due to the orthochromatic component of the nucleus and cytoplasmic basophily, which had to be eliminated through image elaboration. Because of its subjective nature, this operation may in turn lead to further errors. The choice of Astra Blue as an alternative to Toluidine Blue in densitometric analysis of mastocytes is based on its property to restrict staining to the granules of mast cells. A comparison between Astra Blue and the two Toluidine Blue solutions showed that, at all transmission levels, preparations stained with Astra Blue captured more pixels than those stained with Toluidine Blue. Consequently our results suggest that the most suitable technique for densitometric image analysis is fixation of mast cells in IFAA followed with Astra Blue.