Streptavidin biotin immuno peroxidase method

After FeCl₃-induced injury, the occluded carotid artery was removed and embedded in paraffin. Paraffin sections were stained with biglycan-specific antibody (biglycan polyclonal rabbit antibody, abcam, Cambridge, United Kingdom, # 49701, 1:100) following the streptavidin–biotin immuno-peroxidase method (Dako, Glostrup, Denmark). Sample slices of healthy carotid arteries were prepared from paraffin-embedded samples, dewaxed and dehydrated, blocked (5% goat serum, 0.1% BSA and 0.3% triton (Sigma-Aldrich, Burlington, MA, USA, # T8787-100)), and incubated with antibodies against collagen I (rabbit anti-mouse collagen I antibody, Thermo Fisher Scientific Inc., Waltham, MA, USA, # PA1-85319) and collagen IV (rabbit polyclonal collagen IV antibody, abcam, Cambridge, United Kingdom, # ab6586). Then, secondary antibody incubation was performed for 1 h (Alexa Fluor™ 555 goat anti-rabbit IgG (Thermo Fisher Scientific Inc., Waltham, MA, USA # A21428)), and cell nuclei were stained with DAPI (Roche Diagnostics, Mannheim, Germany, final concentration 1.6 µg/mL) and inundated with FluoromountTM (Sigma-Aldrich, Burlington, MA, USA). The documentation was done after drying at 4 °C overnight with microscope Axio Observer.D1 (Carl Zeiss Microscopy GmbH, Oberkochem, Germany, AxioCam MRm, objective LD Plan-Neofluar 40× 0.6 Korr. Ph2 M27).

Twenty four hours after ischemia/reperfusion hearts were taken and paraffin sections of these hearts were stained either with Hematoxylin/Eosin (HE) solution (Sigma, Darmstadt Germany) or immune cell specific with a Streptavidinbiotin-immunoperoxidase method (Dako, Darmstadt, Germany). By HE-staining the total number of cells migrated into the infarcted area of the heart was counted per visual field and data are shown per mm². For immune cell specific analysis paraffin-embedded heart sections were stained with an anti-Ly6G antibody for neutrophil staining (BD Pharmingen, Heidelberg, Germany) and an anti-Mac3 antibody for monocyte staining (BD Pharmingen, Heidelberg, Germany). From 12 areas of the infarct border zone either Mac3- or Ly6G-positive cells were counted and the data are shown per mm².
For the analysis of α-SMA positive cells in the infarct area 21 days after ischemia/reperfusion, α-smooth muscle actin (α-SMA) staining of paraffin sections was performed using anti-α-SMA antibody (Abcam, Cambridge, United Kingdom), anti-rabbit horseradish peroxidase as second antibody (Santa Cruz, Frankfurt am Main, Germany) and Diaminobenzidine (DAB) reagent (DAKO, Darmstadt, Germany) as chromogen.