Anti s100b sh b1

S100B secretion was measured by an enzyme-linked immunosorbent assay, as previously described^{58 (link)}. Briefly, 50 µL of extracellular medium from slices and 50 µL of Tris buffer were incubated for 2 h on a microtiter plate previously coated with monoclonal anti-S100B (SH-B1; Sigma-Aldrich). Next, the samples were incubated with polyclonal anti-S100B (Dako) for 30 min, and then, peroxidase-conjugated anti-rabbit antibody (Amersham) was added for a further 30 min incubation period. A colorimetric reaction with o-phenylenediamine (Sigma-Aldrich) was observed at 492 nm. Results are expressed as percentages of the non-infection control value.

Lipopolysaccharide from Escherichia coli (LPS, 055:B5), TRI Reagent, minocycline, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), metformin, oxamic acid, MCC950, fluorocitrate, PFK1 activity assay, anti-S100B (SH-B1), 4-(2-hydroxyethyl) piperazine-l-ethanesulfonic acid (HEPES), o-phenylenediamine (OPD), and HRP- conjugated anti-goat IgG and anti-p38 MAPK were purchased from Sigma (Saint Louis, MO, USA). Arundic acid was purchased from TOCRIS (Bristol, United Kingdom). Standard GFAP was from Calbiochem (San Diego, CA, USA). Polyclonal anti-S100B and polyclonal anti-GFAP were purchased from DAKO (Carpinteria, CA, U.S.A.). The lactate and lactate dehydrogenase (LDH) assays were purchased from BioClin, Brazil. Polyclonal anti-EAAT2 (GLT1) and anti-EAAT1 (GLAST) were purchased from Abcam (Cambridge, MA, USA) and anti-GLUT1, anti-COX2, anti-TLR4 and anti-RAGE were purchased from Santa Cruz Biotechnology (Inc., Dallas, Texas, USA). Monoclonal anti-Iba1 was purchased from Merck/Millipore (Darmstadt, Germany). Anti-phospho p38 MAPK, anti-Akt and phospho Akt (Ser473) were purchased from Cell Signaling Technology (Danvers, Massachusetts, U.S.A.). Anti-HRP conjugated actin was purchased from Proteintech (Rosemento, IL, USA). Finally, HRP-conjugated anti-rabbit IgG and anti-mouse IgG were purchased from GEHealthcare (Little Chalfont, United Kingdom).

S100B content in the supernatant was measured by ELISA, as described previously [51 (link)]. Briefly, 50 μl of sample plus 50 μl of Tris buffer were incubated for 2 h on a microtiter plate that was previously coated with monoclonal anti-S100B SH-B1 (Sigma-Aldrich, St. Louis, MO, USA). Polyclonal anti-S100 (Dako, Carpinteria, CA, USA) was incubated for 30 min, and peroxidase-conjugated anti-rabbit antibody was then added for a further 30 min. The color reaction with OPD was measured at 492 nm. The standard S100B curve ranged from 0.02 to10 ng/ml and data expressed as nanograms per milligram or nanograms per milliliter. Results are shown as percentages of the control.