5 lipoxygenase

All stainings were done essentially as described [18 (link)] using the mouse monoclonal antibody ED1 (to stain macrophages and activated microglia; Serotec, Germany), rabbit polyclonal antibodies against CD3 (to stain T cells; NeoMarkers, Fremont, USA), rabbit polyclonal antibodies against AQP4 (to stain astrocytes; Sigma, Germany), rabbit polyclonal or mouse monoclonal antibodies against glial fibrillary acidic protein (GFAP; from Dako, Denmark, or NeoMarkers, respectively), anti-human immunoglobulin (biotinylated donkey; polyclonal; Amersham, UK), anti-complement C9 (rabbit polyclonal [21 (link)]), anti-Rab5c (goat polyclonal; Santa Cruz), anti-5-lipoxygenase (rabbit monoclonal; Cell signaling), and anti-Ptpn6 (rabbit polyclonal, Abnova). While the AQP4-specific antibodies could be used without antigen retrieval, the other antibodies required heat-mediated antigen retrieval by steaming the sections for 60 minutes in 50 μM EDTA pH 8.5 (ED1, antibodies against CD3, GFAP, Rab5c, 5-lipoxygenase and Ptpn6), or a treatment for 15 minutes at 37°C with 0.03% Proteinase Type XXIV (Sigma) (antibodies against human immunoglobulin and against complement C9).

All solvents used were HPLC grade; CH₃CN and MeOH for HPLC were purchased from Merck (Darmstadt, Germany). Formic acid (85% v/v) was provided by Carlo Erba (Milan, Italy). Water was purified by a Milli-Qplus system from Millipore (Milford, MA, USA).
Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), BHT (butylated hydroxytoluene), ascorbic acid, quercetin, DPPH (1,1-diphenyl-2-picrylhydrazyl radical), 5-lipoxygenase, linoleic acid, AAPH [2,2^I-azobis(2-amidinopropane) dihydrochloride] and fluorescein sodium salt were purchased from SIGMA (Milano, Italy).