Figure 6

Stimulus-dependent expression of RAGE and ICAM-1 in cremaster muscle venules. Immunostaining was conducted to assess endothelial expression of RAGE (A, C, E) and ICAM-1 (B, D, F) in postcapillary venules of cremaster muscles obtained directly postmortem (Unstimulated, upper panel), after exteriorization and 20 min superfusion (Trauma, middle panel), or after 3 h of TNF-α-stimulation and following exteriorization (TNF-α, lower panel; at least 3 mice/group). Application of primary antibody was performed i.v. before harvesting the cremaster muscle in order to stain RAGE and ICAM-1 on the endothelial surface. Biotinylated secondary antibody, peroxidase-conjugated streptavidin and diaminobenzidine (DAB) were used to detect endothelial expression of ICAM-1 and RAGE as brown signal. Counter-staining was performed by Mayer's hemalaun. Reference bar for the representative images is shown in A and represents 20 μm. Intensity of venular anti-RAGE and anti-ICAM-1 immunostaining during trauma- and TNF-α-induced inflammation were analyzed semiquantitatively and presented as mean ± SEM (G; 0 = no, 1 = weak, 2 = medium, 3 = strong signal). Significant differences (p < 0.05) of TNF-α-induced RAGE or ICAM-1 expression vs. trauma are indicated by the asterisk or the pound key, respectively. Relative ratios of expression of RAGE to ICAM-1 are quantified for the trauma and TNF-α model as illustrated.