Berghman L R, Grauwels L, Vanhamme L, Vandesande F
Laboratory for Neuroendocrinology and Immunological Biotechnology, Zoological Institute, Leuven, Belgium.
J Immunol Methods. 1994 Feb 10;168(2):197-202. doi: 10.1016/0022-1759(94)90055-8.
A technically simple modification of routine (non-adsorbent) multi-well plates, permitting the simultaneous immunocytochemical processing of hundreds of free-floating sections is described. The adaptations consist of (1) making a 1.5 mm wide perforation in the bottom of each well of the multi-well plate, (2) placing a 6 mm wide 50 microns mesh nylon filter on the bottom of each well and (3) preincubating the plate with excess inert protein in order to prevent adsorption of protein reagents. During the incubation of the floating sections with the immunocytochemical reagents, the fluid is retained in the well by capillarity, provided the detergent concentrations within the well do not exceed 0.005% (v/v). The wells can be emptied simply and quickly by blotting the plate bottom with a piece of laboratory paper toweling: the fragile sections are gently caught on the filter, without the risk of loss or damage. Sections start floating again as soon as the next reagent is added to the well. The present method drastically reduces the time needed for rinsing and reagent exchange, making immunocytochemistry on free-floating sections feasible as a primary screening method during hybridoma production.
本文描述了一种对常规（非吸附性）多孔板进行的技术上简单的改进方法，该方法可允许同时对数百个游离切片进行免疫细胞化学处理。改进措施包括：（1）在多孔板每个孔的底部制作一个1.5毫米宽的穿孔；（2）在每个孔的底部放置一个6毫米宽、50微米网孔的尼龙滤膜；（3）用过量的惰性蛋白对多孔板进行预孵育，以防止蛋白质试剂吸附。在用免疫细胞化学试剂孵育游离切片的过程中，只要孔内洗涤剂浓度不超过0.005%（体积/体积），液体就会通过毛细作用保留在孔中。通过用一张实验室纸巾轻拍板底，就可以简单快速地排空孔内液体：脆弱的切片会被轻轻地截留在滤膜上，而不会有丢失或损坏的风险。一旦向下一个孔中加入试剂，切片就会再次漂浮起来。本方法大大减少了冲洗和试剂更换所需的时间，使得对游离切片进行免疫细胞化学作为杂交瘤生产过程中的一种初步筛选方法变得可行。
