Rabbit anti p camk 2

2-, 4- and 6-month EFAD mice were anesthetized with sodium pentobarbital (50 mg/kg), transcardially perfused with ice-cold PBS, brains removed and dissected into cortex and hippocampus, snap-frozen in liquid nitrogen and stored at -80°C, as previous described [133 (link)]. Dissected brains were homogenized in lysis buffer [90 (link),132 (link)] (50 mM Tris-HCl, 150 mM NaCl, pH7.4, 1% Triton X-100, 1x protease inhibitor cocktail) and 40 μg of total protein (BCA protein assay kit; Pierce, Rockford, IL) was separated on 4–12% gradient Bis-Tris gels (Invitrogen) under reducing conditions, and transferred to PVDF membranes [47 (link)]. The following primary antibodies were used: rabbit anti-PSD95 (1:3000, Abcam), mouse anti-synaptophysin (1:2000, Abcam), mouse/rabbit anti-β-actin (1:2000; Abcam), rabbit anti-drebrin antibody (1:1000; Abcam), rabbit anti-NMDAR1/anti-NMDAR2B (1:1000; Millipore), anti-NMDAR2A (1:500; Millipore), mouse anti-apoE (1:600; Santa Cruz), rabbit anti-BDNF (1:200; Santa Cruz), rabbit anti-p-CaMK-II (1:1000; Santa Cruz) and rabbit anti-p-CREB (1:1000; Cell Signaling) [90 (link),132 (link)]. HRP-conjugated secondary antibodies, enhanced chemiluminescence (Amersham, Piscataway, NJ) and Image J software were used to quantify densities of the immunoreactive bands relative to β-actin.

Fixed hatching and first lorica larvae were washed three times for 5 min in PTw to remove sodium azide, and perforated afterwards with a thin needle to allow antibody penetration through the larval cuticle. Perforated larvae were transferred into PBS with 0.5% Triton X-100 (PTx) for permeabilization for 2 h at room temperature, and subsequently blocked in 1% bovine serum albumin (BSA) in PTx for 2 h at room temperature. Before adding the primary antibody, larvae were blocked in 10% normal goat serum (NGS) in PTx twice for half an hour. The analysed primary antibodies (mouse anti-acetylated tubulin (Sigma, #T6793), mouse anti-tyrosinated tubulin (Sigma, #T9028), rabbit anti-pCaMKII (Santa Cruz Biotechnology, #sc-12886), rabbit anti-serotonin (Sigma, #S5545) and rabbit anti-FMRFamide (Immunostar, #20091)) were diluted 1 : 100 in 10% NGS in PTx and incubated for approximately 40 h at 4°C. Continuous washes in 1% BSA in PTx for approximately 7 h to remove the primary antibody were followed by blocking in 10% NGS in PTx twice for half an hour and incubation in Alexa-conjugated secondary antibody diluted 1 : 250 in 10% NGS in PTx for approximately 40 h at 4°C. Finally, secondary antibodies were washed out in PTx, and if needed nuclei were counterstained with Sytox Green.