Mouse anti inos

Platelets samples were lysed (NaCl 0.15 M, Tris 10 mM pH 8, EDTA 0.1 mM, Triton X-100 1%, and protease inhibitor cocktail (ROCHE)) and protein quantification was performed using BCA kit (Thermo Scientific). Protein extracts (10 µg) from platelets from 4 patients and 4 healthy volunteers were prepared with sample buffer containing β-mercaptoethanol, separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred into nitrocellulose membrane. The membranes were blocked with Tris-buffered saline (TBS) supplemented with 0.1% Tween 20 (TBS-T) plus 5% milk for 1 hour and incubated with primary antibodies mouse anti-iNOS (BD610333) and mouse anti-human β-actin (Sigma A1978).

Three days following TBI, mice were anesthetized with Ketamine/Xylazine (100/10 mg/kg, intraperitoneally) and then transcardially perfused with 1× phosphate buffer saline (PBS) followed by 4% paraformaldehyde (PFA). Mice brains were dissected, placed in 4% PFA for 24 h, and paraffin-embedded, and 8 µm sections were made using a microtome and used for the staining. The staining procedure started with deparaffinized brain sections in different gradients of xylene/ethanol, followed by antigen retrieval in a pressure cooker for 15 min, washing with 1× PBS, and blocking in 5% BSA for 2 h at room temperature. The following primary antibodies were added subsequently and incubated overnight at 4 °C: mouse anti-iNOS (1:200, Sigma), and rabbit anti-3NT (1:200, Cell Signaling). After washing with 1× PBS, slides were incubated with secondary antibodies for 1 h at room temperature (Taxes red labeled donkey anti-rabbit IgG and Alexa Fluor donkey anti-mouse IgG) (1:1000; Jackson, Immunoresearch, West Grove, PA, USA). Then, the slides were washed with 1× PBS, mounted with ProLong^® Gold Antifade Mountant with DAPI (Molecular Probes), and images were captured using fluorescence microscopy. Fluorescence intensity was analyzed using ImageJ software (NIH, USA).

Deparaffinized mice brain sections, followed by antigen retrieval process for 15 min in a pressure cooker, washed three times with 1× PBS for a duration of 5 min and blocked with 5% BSA dissolved in TBST for another 2 h. on a rocker. After that, the primary antibodies were added and incubated overnight at 4 °C, 1:1000; mouse anti-cofilin (Abcam), 1:1000; rabbit anti-IBA1 (Wako, Japan), rabbit anti-3NT (1:200, Cell Signaling), mouse anti-iNOS (1:200, Sigma). After 24 h, the slides were washed three times with 1× PBS, and the secondary antibodies were added and incubated in the dark container for 1 h. at room temperature Alexa Fluor donkey anti-mouse IgG (1:1000; Jackson, Immunoresearch, West Grove, PA, USA), (Taxes red labeled donkey anti-rabbit IgG (1:1000; Jackson, Immunoresearch). The slides underwent a series of 3 washes using 1× PBS and were mounted with DAPI (Molecular Probes, Eugene, OR, USA). The images of the localization of cofilin in microglia and iNOS with 3NT were acquired using fluorescent microscopy. Cofilin fluorescence intensity, rod fluorescence intensity, and rod length around the hemorrhagic region were measured using ImageJ software 1.53t (NIH), and the number of microglia was determined by counting the activated cells around the hemorrhage using ImageJ software 1.53t (NIH).