Immunology and Infection
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观察活胰腺组织切片中的胰岛功能和胰岛-免疫细胞相互作用
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Summary April 12th, 2021
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本研究介绍了活胰腺组织切片在胰岛生理学和胰岛免疫细胞相互作用研究中的应用。
Transcript
我们的方案将有助于回答免疫细胞相互作用对胰岛功能的影响。活胰腺组织切片方法允许研究胰岛的生理学和功能，同时保持潜在的病理和天然微环境。这种方法可以提供对胰腺疾病的疾病过程的见解，例如胰腺炎，I型糖尿病和II型糖尿病。
首先，将块排列在试样夹具座上，使其不超过刀片宽度，确保当振动器缓慢移动时，刀片可以向前移动尽可能短的距离。在试样支架上涂上一行超级胶水，使用胶水分配器的末端形成薄层，然后将组织块翻转到胶水上，使靠近组织的一面朝上。轻轻按下块，让胶水干燥三分钟。
接下来，用螺钉将板连接到振动器上，并调整刀片高度和行进距离，以便刀片在块的长度上移动，并且勉强高于它们。用镊子轻轻轻轻推块以检查胶水是否干燥，然后用冷却的细胞外溶液填充振动盘，直到刀片被覆盖。将振动切片机设置为120微米厚的切片并启动它，然后观察从组织块上脱落的切片。
当切片从块上漂浮时，将画笔或镊子放在它们下面，然后将切片放在含有三毫摩尔D-葡萄糖和胰蛋白酶抑制剂的Krebs-Ringer碳酸氢盐缓冲液中。将含有切片的板放在摇臂上，并在室温下以25 RPM孵育一小时。如果切片需要保存更长时间，请将切片置于15毫升切片培养基中，并将平板置于培养箱中。
将准备好的切片在37摄氏度下孵育以进行当天研究，或在实验前将24摄氏度培养过夜的切片转移到37摄氏度至少一小时。在记录前至少一小时打开显微镜，并将载物台顶部培养箱平衡至37摄氏度。将盖板玻璃底培养皿固定在舞台上，其中包含切片。
通过在明场模式下设置10X物镜来聚焦显微镜，并通过在切片中寻找橙棕色的椭圆形来使用明场模式定位小岛。通过按下显微镜触摸屏控制器上的CS按钮，将显微镜切换到共聚焦成像。要使用反射率查看胰岛，请打开 638 激光探测器和 PMT 探测器。
将激光功率设置为1%至2%，然后关闭陷波滤波器。使用405纳米激光和PMT检测器观察核酸染色。混合检测器用于CD8抗体检测和488纳米激光和混合检测器，用于观察胰岛素四聚体。
使用载物台控制器的 X 和 Y 旋钮将感兴趣的小岛居中置于视野中。找到感兴趣的小岛后，切换到20倍物镜并放大，使小岛占据大部分帧。取一个z-stack的胰岛，然后找到细胞活着的最佳光学切片，并且任何周围的免疫细胞都处于焦点中。
将显微镜设置为以XYZT模式记录，并优化设置以在几个小时内每20分钟记录一次所选步骤的z-stack。使用明场和反射光显微镜观察胰腺组织切片中的胰岛。胰岛中的胰岛素增加了颗粒度并导致大量反射光的吸收，增加了胰岛的可见性。
通过染色评估活人胰腺组织切片的活力。活细胞以绿色表示，死细胞以蓝色表示。生存能力的另一个积极指标是可观察到的基础活性。
当活的胰腺切片加载细胞可渗透的钙指示剂时，在小鼠和人胰腺切片中观察到葡萄糖刺激。还量化了葡萄糖刺激期间单个细胞的荧光。使用地噻嗪染色和反射光显微镜观察完整和患病的胰岛。
由于免疫细胞浸润和细胞死亡，患病胰岛开始失去颗粒。免疫细胞群可以使用CD8抗体和胰岛素四聚体染色来鉴定。共染色表明这些细胞是特异性靶向胰岛素抗原的效应性T细胞。
在此过程中要记住的最关键的事情是始终将切片保留在溶液蛋白酶抑制剂中。遵循该程序，可以执行免疫细胞实验中的许多功能，从而允许研究免疫细胞相互作用和胰岛功能的影响。
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