Effect of TTC Treatment on Immunohistochemical Quantification of Collagen IV in Rat Brains after Stroke.

Abstract

Although used extensively in stroke research, there is limited knowledge of how 2, 3, 5-triphenyltetrazolium chloride (TTC)-treated rat brain sections are altered and if they can be used for immunohistochemical quantification after staining with TTC. In the present study, we hypothesized that TTC treatment (TTC+) would not interfere with collagen IV immunohistochemical staining compared with non-TTC-treated (TTC-) brain slices. We further hypothesized that there would be no difference in autofluorescence or nonspecific secondary antibody fluorescence between TTC+ and TTC- brain slices. Coronal brain sections of male Wistar rats (n = 5/group) were either treated with TTC or not after middle cerebral artery occlusion or sham surgery, and processed for immunohistochemical staining with mouse anti-collagen IV as the primary antibody, and goat anti-IgM as the secondary antibody. Four images were taken in the cerebral cortex of the contralateral side of infarction in each brain slice using an Olympus BX50 fluorescence microscope, and average intensity of the entire image was quantified using the Metamorph software. Compared with TTC- brain slices, TTC+ brain slices showed a significantly lower autofluorescence (P < 0.05), but was unchanged for nonspecific secondary antibody fluorescence. In addition, TTC+ brain slices had similar collagen IV staining intensity compared with TTC- brain slices. These results demonstrate that TTC+ brain slices are usable for immunohistochemical quantification.