Dab enhanced liquid substrate system

Immunohistochemical (IHC) analysis of α-SMA was performed with mouse monoclonal antibody against α-SMA. Following deparaffinization and rehydration, 0.01 M boiled citrate buffer (pH 6.0) was added into each section, and heated at 97 ± 1 °C for 10 min to achieve antigen retrieval. The sections were cooled to room temperature (RT), followed by incubation with 3% H₂O₂ (1% in 0.01 M PBS, v/v) for 10 min to remove endogenous peroxidase. After placing in PBS and blocking with 5% albumin bovine serum (Sigma-Aldrich, MO, USA) at 37 °C for 30 min, the sections were exposed to the primary antibody mouse anti-α-SMA (1:3000; Abcam, Cambridge, UK) at 4 °C overnight. Subsequently, the sections were pre-warmed to RT for 30 min and incubated at 37 °C for 30 min. After rinsing in PBS, the sections were exposed to the secondary antibody Envision System-HRP-conjugated anti-mouse IgG (DAKO, Denmark) at 37 °C for 40 min. After the final wash, the sections were stained with 3,3′-diaminobenzidine (DAB) and recorded using a DAB-enhanced liquid substrate system (DAKO). Lastly, the sections were counterstained with hematoxylin. To verify the specificity of IHC assay, the primary antibody was replaced by Mouse (G3A1) mAb IgG1 Isotype Control (Cell Signaling Technology, MA, USA) as a negative control. The densitometry analysis of positive staining was conducted with Image-Pro Plus tool.

Coronal brain sections (5 μm thick) were randomly selected between 0 and 2.0 mm posterior to the bregma. After deparaffinization and rehydration, the sections were incubated for 10 min at 95–98°C in 0.01 M citrate buffer (pH 6.0) for antigen retrieval. The sections were allowed to cool down to room temperature (RT) and were then incubated for 15 min in H₂O₂ (1% in 0.01 M PBS, v/v) and 0.5 hours in 5% bovine serum albumin (Sigma, St. Louis, USA) blocking solution (5% in 0.01 M PBS, w/v) at RT. Then, the sections were incubated overnight at 4°C in a solution of anti-NeuN antibody (1 : 500, v/v, clone A60, MAB377, Merck Millipore, Darmstadt, Germany). The sections were washed with 0.01 M PBS and then incubated with the Mouse EnVision+ System-HRP (K4006, DAKO, Glostrup, Denmark) for 1 hour at RT. Positive staining was visualized with 3,3′-diaminobenzidine (DAB) using a DAB-enhanced liquid substrate system (DAKO, Glostrup, Denmark). Finally, the sections were counterstained with hematoxylin, dehydrated, mounted, and observed under a microscope (Leica DM4000 BLED, Wetzlar, Germany). The number of immunopositive cells was counted in five randomly selected fields from the peri-infarct area per slice and presented as the number of cells/mm². All of the positively stained cells (brown color) were included in the count regardless of their morphology.