Double immunostaining of microglial cells with its membrane-bound receptor TLR
after spinal cord ischemia reperfusion (I/R) injury. (A) Representative micrographs show the cellular location of Toll-like receptor (TLR4; red) with antibodies against microglial specific marker (Iba-1; green) at 12 h and 36 h after I/R injury. Arrows delineate co-localization. Scale bars are 100 μm. (B) Histogram for quantification of co-localized cells (cells with yellow signals). (C) Quantification of the TLR4 immunoreactivity is presented as average of three fluorescence intensity (FI) of three independent experiments. Double immunohistochemistry showed TLR4 was highly expressed on spinal microglial after I/R injury. The rats pretreated with minocycline, TAK-242, PDTC were suggested significantly decreased TLR4 immunoreactivity after I/R and the number of TLR4-Iba-1 positive microglia, whereas above effects synthetically increased in rats receiving LPS. All data are presented as mean ± SEM (n = 8 per group). **P < .01 compared to Sham group; ##P < .05 compared to I/R group.