The dilu tion series of sodium nitrite was utilized to make the nitrite standard reference curve. Western blot analysis After treating cells with cytokines and LPS, cells were washed twice with ice cold phosphate buffered saline and harvested in lysis buffer containing 50 mM Tris HCl, one mM EDTA, a hundred mM NaCl, 0. 1% SDS, one mM PMSF, 1 mM sodium orthovanadate, one ug/ml leu peptin, one ug/ml pepstatin, and ten ug/ml aprotinin. The extract was centrifuged at 10,000 ? g for 15 minutes at 4 C for you to eliminate cell debris. Protein concentra tion was established by utilizing a BCA protein assay kit according to the suppliers guidelines. Equivalent amounts of professional tein for every sample were resolved in 12% Tri cine SDS Page at 120 V in duplicates. Immediately after electrophoresis, proteins were transferred to 0. two um PVDF membranes at 250 mA for two h. Membranes were incubated in Tris buffered saline, pH 7.
four with 0. 1% Tween 20 containing 5% non excess fat milk for 1 h at space temperature. The blots have been then incubated with sPLA2 IIA polyclonal antibody overnight at 4 C. Just after washing with TBS T, blots inhibitor SB-715992 were incubated with goat anti rabbit IgG horseradish peroxidase for one h at room temperature. The blots have been then washed three times with TBS T. Immu nolabeling was detected by chemiluminescence. For loading control, the blots selleckchem were reacted with monoclonal anti b actin peroxidase. For quantification, blots had been scanned as well as the intensity of protein bands was measured as optical den sity employing the Amount One program. sPLA2 IIA bands had been detected at 15 kDa. Ratios of sPLA2 IIA to b actin had been calculated for every sample. Immunohistochemistry DITNC cells and key astrocytes had been plated onto poly L lysine coated glass coverslips. Right after treatments, cells were fixed in 4% paraformaldehyde in PBS for 15 min at space temperature.
Immediately after washing 3 times with PBS, samples were incubated for 10 min with PBS containing 0. 5% Triton X 100. Nonspecific binding of antibodies was blocked by 5% normal goat serum for 1 h at area temperature. Cells have been then incubated overnight at 4 C in 0. 5% NGS with anti sPLA2 IIA polyclonal antiserum, anti GFAP monoclonal

antibody for astrocytes, or anti CD11b antibody for microglial cells. The cells have been washed with PBS and incubated for one h at room temperature with fluorescein isothiocyanate labeled goat anti mouse and Texas red labeled goat anti rabbit secondary antibody, and finally washed again with PBS. Cells have been incubated for 10 min with Hoechst 33342 being a counter stain for nuclei. Cover slips had been then mounted onto microscope slides and fluorescent intensity measurements had been performed at area tem perature making use of the Olympus X 41 fluorescence micro scope and 40? objective lens. For immunofluorescence staining of F actin, BV 2 cells in cover slips had been fixed with 4% paraformaldehyde for 20 min and permeabilized by 0.