论文：融合共对焦高倍显微镜、dSTORM 和质谱技术设备证明与微米机构脂质媒体在血脑天然屏障杀手穿越历程中关联的蛋白酶质冠层的演变成链接搜索：//pubs.rsc.org/en/content/articlehtml/2022/nr/d2nr00484d小说作品：Matteo Battaglini ORCID ，Natalia Feiner bc， Christos Tapeinos a，Daniele De Pasquale a， Carlotta Pucci a，Attilio Marino a，Martina Bartolucci d，Andrea Petretto d，Lorenzo Albertazzi bc和 Gianni Ciofani 节选：材料和方式方法LMNV制法脂质吸引力微米质粒载体 (LMNV) 的生产制造服务协议改编歌曲自让我们司专班原来的学习，融入了热超声清洗波和油田均质化 (HPH) 的方式。20简来之，让我们司将差异的脂质交织在来，分为 2.5 mg 油酸（Sigma-Aldrich）、25 mg 1-硬脂酰-rac -甘油（Sigma-Aldrich）、2.5 mg 油酸（Sigma-Aldrich）、2.5 mg 1,2-二棕榈酰-rac-甘油-3-磷酸胆碱（Sigma-Aldrich）和 4 mg 1,2-二硬脂酰-sn-甘油-3-磷酸乙酸乙酯胺与共轭甲氧基聚乙二醇 (mPEG-DSPE) (5000 Da, Nanocs)，及其 84.5 μl 超顺吸引力被氧化铁微米离子 (SPION) 的乙酸乙酯水溶液 (3 nm 孔径，15 wt%；英国学习微米装修材料司) 放进 6 ml 夹层玻璃小瓶中。将3 ml加温（70 °C）的Tween® 80 (Sigma-Aldrich) 稀硫酸 (1.0 wt%) 引入脂质/SPION单一体中，并施用mri波摄像头 (Fisherbrand™ Q125 Sonicator) 实现mri操作1五1分的时间（波动30%，工作功率120 W）。mri操作后，施用均质机以100 [细分隔符（1/6-em）]000 psi的压为对相混物实现各类高压力均质操作（共实现5次各类高压力均质操作）。納米膜蛋白在4 °C时以16 [细分隔符（1/6-em）]000 g抽滤式901分的时间（共实现3次）实现纯化，然而重单一于水面。为了能实现共自动对焦显像，LMNV 施用荧光 Vybrant DiO 癌细胞图标有机染料（Invitrogen）实现图标。将 5 mg 納米膜蛋白与 20 μM DiO 在 37 °C 下孵育 2 个小时，然而在 4 °C 时以 16 [细分隔符（1/6-em）]000 g抽滤式 90 1分的时间（三四次）实现洗衣。对间接随机性光学玻璃复建高倍显微镜 (dSTORM) 研究分析，在制取历程上将 3 mg mPEG-DSPE（5000 Da，Nanocs）与 1 mg DSPE-PEG-Cy3（5000 Da）相混实现染料。Materials and methodsLMNV preparationThe protocol for the fabrication of lipid magnetic nanovectors (LMNVs) was adapted from previous works of our group combining hot ultra-sonication and high-pressure homogenization (HPH) methods.20 Briefly, we mixed different lipids including 2.5 mg of oleic acid (Sigma-Aldrich), 25 mg of 1-stearoyl-rac-glycerol (Sigma-Aldrich), 2.5 mg of oleic acid (Sigma-Aldrich), 2.5 mg of 1,2-dipalmitoyl-rac-glycero-3-phosphocholine (Sigma-Aldrich), and 4 mg of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine with conjugated methoxyl poly(ethylene glycol) (mPEG-DSPE) (5000 Da, Nanocs) with 84.5 μl of an ethanol solution of superparamagnetic iron oxide nanoparticles (SPIONs) (3 nm diameter, 15 wt%; US Research Nanomaterials Inc.) into a 6 ml glass vial. 3 ml of pre-warmed (70 °C) Tween® 80 (Sigma-Aldrich) solution (1.0 wt%) were added to the lipid/SPION dispersion and sonicated using an ultrasonic tip (Fisherbrand™ Q125 Sonicator) for 15 min (amplitude 30%, 120 W). After the sonication, the mixture underwent high-pressure homogenization with a homogenizer at 100[thin space (1/6-em)]000 psi (5 passages of high-pressure homogenization were performed). The nanovectors were purified by centrifugation at 16[thin space (1/6-em)]000g for 90 min at 4 °C (three passages) and then re-dispersed in water. For confocal imaging, LMNVs were labeled with the fluorescent Vybrant DiO cell-labeling dye (Invitrogen) by incubating 5 mg of nanovectors with 20 μM of DiO for 2 h at 37 °C and then washing them by centrifugation at 16[thin space (1/6-em)]000g for 90 min at 4 °C (three passages). For Direct STochastic Optical Reconstruction Microscopy (dSTORM) analysis, the staining was obtained by mixing 3 mg of mPEG-DSPE (5000 Da, Nanocs) with 1 mg of DSPE-PEG-Cy3 (5000 Da) during the preparation procedure.

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