Immunohistochemical detection and quantification of T cells in the small intestine of Isospora suis-infected piglets--influence of fixation technique and intestinal segment.

Quantification of immunohistochemical results constitutes an important tool in the analysis of cells and tissue that is not readily replaced by other techniques. For reliable quantification, it is essential to consider factors such as tissue fixation and tissue sampling. We report a study on the model of the intestine of Isospora suis-infected piglets, in which we addressed (1) whether the quantity of detectable T cells in the intestinal mucosa is the same in formalin-, HOPE®-, and cryo-conserved material or whether the amounts of T cells at least correlate with one another; and (2) whether single jejunal segments differ in regard to the quantity of mucosal T cells and variability of lymphocyte infiltration. Quantification of T cells in histological sections of different parts of the jejunum of 15-22 day old piglets infected with I. suis was performed using an anti-CD3-antibody and stereological point counting. Area fractions of T-cell profiles per intestinal mucosa profile were higher in cryo-conserved samples than in HOPE®- and formalin-conserved material but no correlation between different fixations could be found. The proximal part of the jejunum contained fewer T cells compared with mid- and end-jejunum. Coefficients of variation did not differ between the intestinal segments. For quantification of T cells in the gut mucosa of piglets infected with I. suis, the cryo-conserved mid jejunum seems most suitable in cases when unbiased sampling of the complete intestine is not feasible. It is generally not possible to compare quantitative results of immunostaining in samples conserved by different methods.