The primer sequences more info used in this study were as follows, NF Inhibitors,Modulators,Libraries B p65, The SYBR green PCR master mix was used for real time PCR analysis. The relative differences in expression between groups Inhibitors,Modulators,Libraries were expressed using cycle time values normalized with b actin, and relative differences between control and treatment groups were calculated and expressed as relative increases setting control as 100%. Immunohistochemistry Mouse brains were fixed with 4% paraformidehide in Phosphate Buffered Saline and processed for immunostaining as described previously. Human postmortem brains were processed to Paraffin sections for immunohistochemistry. Microglia were stained with rabbit anti Iba1 antibody. Mouse NOX membrane subu nit gp91phox was immunostained with monoclonal anti mouse gp91phox or rabbit polyclonal anti gp91phox IgG.
Human gp91phox was immunostained with goat polyclo nal gp91phox antibody. Caspase 3 was immunostained with polyclonal anti cleaved caspase 3 antibody. Neu rons were stained with Neu N or MAP2 antibody. Astrocytes were labeled with GFAP antibody. Immuno labeling was visualized by using nickel enhanced 3,3 diaminobenzidinne or Alexa Fluor 488 Inhibitors,Modulators,Libraries or 555 or 633 dye. In situ visualization of O2 and O2 derived oxidant production In situ visualization of O2 and O2 derived oxidant production was assessed by hydroethidine histochemis try. Mice were injected with dehydroethidium in 0. 5% carboxymethyl cellulose at 23. 5 hrs after the last dose of ethanol. Brains were harvested 30 min later and frozen sections were examined for hydroethidine oxidation product, ethidium accumu lation, by fluorescence microscopy.
Fluoro Jade B staining with Neu N labeling Brain sections were immunostained with mouse Neu N antibody. Immunolabeling was visualized by using Alexa Fluor 555 dye. Sections were rinsed three times with PBS and one time with water before performing Fluoro Jade B procedure. Sections stained with Neu N were mounted on superfrost plus Inhibitors,Modulators,Libraries microscope slides and air dried overnight. The sections were rinsed in distilled water for 2 min to rehydrate and transferred to a solution of 0. 06% potassium permanga nate for 10 min. The sections were then rinsed in dis tilled water for 2 min and placed in a 0. 0004% Fluoro Jade B solution made by adding 4 ml of a 0. 01% stock solution of Fluoro Jade B to 96 ml Inhibitors,Modulators,Libraries of 0. 1% acetic acid. After 20 min in the Fluoro Jade B staining solution, selleck chem the stained slides were thoroughly washed in distilled water, dehydrated and coverslipped. Microscopic quantification Immunoreactivity of mouse gp91phox and fluorescent intensity of Fluoro Jade B and ethidium were quantified using Bioquant Image Analysis Software. Images were captured on an Olympus BX51 microscope and Sony DCX 390 video camera at 40X.