Quantitative evaluation of different fixatives for Masson trichrome staining in canine intestinal tissue: An image analysis-based comparative study

Neutral buffer formalin (NBF) has been widely used as a fixative for histological staining for ages. However, for some special staining, such as Masson trichrome staining, it has been proposed that the suitable fixative was Bouin’s solution. The purpose of the present study was to compare the different fixatives and post-fixative protocols on the quality of Masson trichrome staining. The canine intestines (n = 10), which were the duodenum and colon, were used as the candidate organs since they comprised of various tissue components. The fixatives and staining protocols were divided into four groups, which were Groups 1 and 2; the tissues were fixed in Bouin’s solution; Groups 3 and 4 were fixed in NBF. In addition, Groups 2 and 4 were post-fixed with Bouin’s solution before Masson trichrome staining. The quantitative staining was evaluated by using Image analysis software (ImagePro plus 6.0). The results clearly showed the differences between post-fixation protocols rather than fixatives used. The strongest blue intensity was observed in the groups with post-fixation, which was omitted (Groups 1 and 3), while the red area was found to be the highest in the groups with post-fixation (Groups 2 and 4). When comparing different fixatives within the same post-fixation protocol, a higher red area was observed in NBF groups. On the other hand, the blue area and the blue intensity were found in the groups with Bouin’s solution as a fixative. In conclusion, the results from this study showed no obvious differences in Masson trichrome staining between different fixatives i.e. NBF and Bouin’s solution. Instead, the post-fixation protocol revealed the significant differences between the parameters studied. Therefore, the essential step for better-differentiated staining was the post-fixation process with Bouin’s solution, as it can increase the distinction between collagen fibers and the other components.