Following a modified version of a previously described protocol [ 30], 100 mg brain tissue were homogenized in 15 mL cold Minimum Essential Medium (MEM) (Gibco, Life Technologies) in a glass tissue grinder (Wheaton), homogenates adjusted to a final concentration of 15% Ficoll and centrifuged at 6000 g and 4°C for 20 min.
Samples from human brain tissue were homogenized in extraction buffer (see blow) by pulsed ultrasonification at 4°C, followed by centrifugation at 10000×g at 4°C for 30 min. The resulting supernatant was used for protein analysis.
CT-2A tumor and contralateral normal brain tissue were homogenized in ice-cold lysis buffer (Cell Signaling Technology, Beverly, MA, USA) containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM NaPPi, 1 mM α-glycerophosphate, 1 mM Na3PO4, 1 µg/mL leupeptin and 1 mM phenylmethylsufonyl fluoride.
Neuronal cells or brain tissue were homogenized in 5 ml of solution A [0.32 M sucrose (34 g/500 ml), 0.5 mM CaCl2 (36 ml/500 ml), 1 mM NaHCO3 (42 mg/500 ml), 1 mM MgCl2 (102 ml/500 ml)] containing protease and phosphatase inhibitors with 12 strokes of a 19×84 mm tissue grinder (Potter Elvehjem, plastic coated) at 800 r.p.m.
Twenty milligrams of brain tissue were homogenized in five volumes of Tris-buffered saline plus (TBS+) buffer (Tris HCl 50 mM pH 7.4, NaCl 175 mM, EDTA 5 mM, PMSF 0.1 mM and N-ethylmaleimide 1 mM; Sigma) using a glass homogenizer on ice.
For determination of CCL2, brain tissue was homogenized (Precellys homogenizer, Peqlab) in the threefold volume (w/v) of ice-cold ELISA blocker (Thermo Scientific) containing protease inhibitors.
This eluate was defined as the nonpolar fraction and was analyzed using HPLC method C. Brain tissue was homogenized with an ultrasonic homogenizer (Braunsonic 1510, Kronberg, Germany) in water, under ice cooling, and subsequently centrifuged at 4,000 rpm for 5 min at 4°C.
The brain tissue was homogenized with a glass homogenizer with the ratio of 100 mg tissue:1 ml ice-cold PBS (pH 7.4) and centrifuged at 12,000 g for 20 minutes at 4°C.
Brain tissue was homogenized in a glass homogenizer with extraction buffer (20 mmol/L Tris-HCl, pH: 7.5, 150 mmol/L NaCl, 1% Triton X-100; 1 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L ethyleneglycoltetraacetic acid, 2.5 mmol/L pyrophosphate, 1 mmol/L β-glycerophosphate) containing protease and phosphatase inhibitor cocktails (Roche Applied Science, Indianapolis, IN, USA).
Briefly, freshly removed brain tissue was homogenized with a glass Teflon homogenizer in isolation buffer (10 mM triethanolamine (TEA), 10 mM acetic acid (HAc), 280 mM sucrose, 0.2 mM EGTA, pH 7.4 with KOH).
Brain tissue was homogenized in 0.2 M ice-cold perchloric acid and the homogenates were cooled on ice for 30 min to deproteinize.
