Computational protocol: Oligomeric amyloid-beta induces MAPK-mediated activation of brain cytosolic and calcium-independent phospholipase A2 in a spatial-specific manner

Similar protocols

Protocol publication

[…] Pulverized cerebrum tissues were homogenized in 1X NP40 on a cooling tissue homogenizer (Cryolys Precellys Evolution Homogenizer). NP40 homogenates were centrifuged at 12,300 rpm for 20 min at 4 °C and supernatants were run into NuPage 4–12% Bis-Tris (Life Technologies, NY) under reducing conditions. Samples were normalized based on total protein content, which was estimated by the BCA protein assay. Western blot analyses were performed using antibodies against cPLA2 (sc-454 and sc-376,636, Santa Cruz Biotechnology -SCB-), phospho-cPLA2 -S505- (2831, Cell Signaling Technology -CST-), iPLA2 (sc-376,563, SCB; and NBP1–81586, Novus), MAPK p42/p44 (4695, CST), phospho-MAPK p42/p44 -T202/Y204- (4370, CST), MAPK p38 (8690, CST), phospho-MAPK p38 (rabbit polyclonal, CST), SAPK/JNK (9252, CST), active JNK (V7931, Promega), PKCα (2056, CST), PKCδ (9616, CST), PKCλ (610,207, BD Biosciences), phospho-PKC pan -βII Ser660- (9371, CST), phospho-PKCα/βII -T638/641- (9375, CST), phosphor-PKCδ -T505- (9374, CST), phospho-PKCζ/λ -T410/403- (9378, CST), CaMK2 (sc9035, SCB), pCaMK2 (sc-12,886-R, SCB), β-Tubulin (2146, CST), GAPDH (MAB374, Millipore), VDAC (4866, CST). Relative intensities were quantified using ImageJ software. [...] Matrix-assisted laser desorption/ionization (MALDI) imaging of fatty acids was carried out as previously described []. Briefly, fresh frozen brain from adult C57BL/6 J WT mouse was cryosectioned at 10-μm thickness. Brain slices were transferred onto the conductive side of indium tin oxide (ITO) slides and desiccated in vacuum for 30–60 min. After drying, N-(1-naphthyl) ethylenediamine dihydrochloride matrix was applied by the Bruker ImagePrep device (Bruker Daltonics, Bremen, Germany). MALDI mass spectra were acquired in the negative ion mode using a reflectron geometry MALDI-TOF mass spectrometer (Ultraflextreme; Bruker Daltonics) equipped with a neodymium-doped yttrium aluminum garnet (Nd:YAG)/355-nm laser as the excitation source. Imaging data were analyzed using FlexImaging v3.0 and BioMap v3.8. Ion images were generated with a bin width of ±0.2 Da. The normalization method was total ion count (TIC). […]

Pipeline specifications

Software tools ImageJ, flexImaging
Applications Mass spectrometry imaging, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Alzheimer Disease, Neurodegenerative Diseases, Neurotoxicity Syndromes
Chemicals Calcium, Fatty Acids, Unsaturated, Lysophosphatidylcholines, Oxygen, Arachidonic Acid