Figure 3. Toll-like receptor 4 (TLR4) mRNA localized to inner stripe of the outer medulla (ISOM) by in situ hybridization.

Wild-type kidneys were studied at 4 h reperfusion. (a) Ischemic antisense. The darkfield photomicrograph shows in situ hybridization for TLR4 mRNA in ischemic kidney using antisense S³⁵ TLR4 mRNA. White dots denote aggregates of silver grains overlying TLR4 mRNA. Red arrows denote increased expression of TLR4 in the vascular bundles of the ISOM of the ischemic kidney. One of these vascular bundles is outlined by a red box that is further studied in d–f. (b) Ischemic sense. This negative control shows the absence of staining of the ischemic kidney hybridized with sense S³⁵ TLR4 mRNA. (c) Sham antisense. This negative control shows the absence of TLR4 mRNA in the non-ischemic kidney. (d) Bright field hematoxylin and eosin. Serial sections of the area delineated by the red box in a were studied. Arrows point to the same vasa rectae in the serial sections d–f. (e) Immunostaining with anti-vWF (von Willebrand’s factor), which stains the endothelium. (f) In situ hybridization for TLR4. White dots indicate in situ hybridization with S³⁵-labeled antisense TLR4 RNA.