13, 14 However, only a few studies have investigated the subcortical GM structures and have found, contrarily to WM, an increase of tissue anisotropy. Numerous studies have applied DTI to characterize microstructural damage in cerebral WM and have demonstrated abnormal low FA and high MD in patients with MS demonstrating a change in myelin architecture. 7, 8 Indeed, volume reduction of deep nuclei has been demonstrated at the earliest clinical stages of MS, 9 ⇓– 11 while cortical GM involvement was identified later in the disease course. Volumetric assessment has become a well-established method for the measurement of GM brain atrophy, which has been shown to progress more rapidly than either WM or whole-brain tissue loss 5, 6 in several cortical regions as well as in subcortical GM structures. Diffuse microscopic alterations and GM tissue loss can, however, be quantified in subcortical GM structures by DTI and volumetric measurement, respectively. However, in vivo detection of GM alterations by conventional clinical imaging techniques is still challenging due to both limited tissue contrast and spatial resolution. TBS = Tris, pH 7.ABBREVIATIONS DD disease duration EDSS Expanded Disability Status Scale FA fractional anisotropy FMRIB Functional Magnetic Resonance Imaging of the Brain GM gray matter ICV intracranial volume MD mean diffusivity MSFC Multiple Sclerosis Functional Composite PD proton attenuation RR relapsing-remitting SP secondary-progressive T2-LL T2-lesion loadĪlthough MS has traditionally been considered a WM disease, pathologic studies dating back to the 19th century, 1, 2 along with more recent reports, 3, 4 have identified GM involvement. Careful attention to maintaining equivalent sample volumes is often the most practical approach. Residual formic acid interferes with most protein assays and protein levels may not be proportional to starting materials in any case. Normalizing between samples is problematic. Neutralized samples should have a final in assay dilution of at least 10- fold. Indicator should be added to the neutralization buffer to signal when pH 7 is reached.Īssay immediately - DO NOT FREEZE neutralized samples (precipitates of highly enriched insoluble hydrophobic species will form that interfere with assay signal).Ĭare should be taken to have equivalent pH for all samples. If dried completely formic acid brain samples form a plastic-like pellet that is highly resistant to resolubilization.Īliquots of the remaining material must be neutralize (to pH 7) with 5 M NaOH in 1M Tris. The bulk of the formic acid buffer needs to be evaporated off (down to 10-20% of original vol) with a nitrogen stream. Steps to prepare formic acid extracts for Aβ assay Normalization between samples is also problematic. HOWEVER, a difficult multi-step process is required to prepare formic acid fractions for assay (both E1 and whole tissue extracts). For mature β-amyloid deposits in human and old mice brain the highest extraction yields are still observed with this buffer. Hence, E1 fractions with SDS should have a final in assay dilution of at least 3-fold.Įarly protocols used formic acid to extract Aβ from AD brain. Biolegend Aβ kits are resistant to SDS and samples with ≤ 0.2% SDS can be used with minimal loss in sensitivity (< 10%). HOWEVER, SDS is not compatible with many ELISA systems. The ionic detergent SDS gives more complete Aβ extraction from insoluble pellets (> 90% with young and ≈ 50% for older mice). Biolegend's Aβ ELISA's (and most other ELISA's) are compatible with non-ionic detergents. mice > 18 months) with heavy mature β-amyloid deposition. Proportion drops rapidly 20% or less in old animals (i.e. Remove supernatant (S1 or soluble fraction) - usually contains 12 months) up to 80% of total mouse brain Aβ is extracted by Triton X-100.Spin for 20 minutes 350,000xg (or equivalent). EDTA 1 (2mM) can be a useful addition to homogenization buffers (see notes below). ![]() ![]() Teflon/glass homogenizer with 5-6 passes on ice. Homogenize in TBS with protease inhibitors (Pierce sells excellent solid tablets for use with homogenization buffers) at 5mLs per 1g tissue.Soluble and insoluble tissue fractionation - This protocol generates soluble and insoluble subcellular fractions for analysis of Aβ partitioning in tissues. Preparation of Brain Samples for LEGEND MAX™ Beta Amyloid ELISA
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