Mähr R, Lezzi M, Eppenberger H M
J Cell Sci. 1977;27:1-12. doi: 10.1242/jcs.27.1.1.
In this paper we describe a method for the rapid mass isolation of polytene nuclei from Chironomus salivary glands. The procedure for the isolation of glands involves 5 principal steps; (a) freezing Chironomus larvae in liquid propane; (b) breaking open frozen animals in a pre-cooled mortar; (c) thawing the fragments in sucrose medium, free of divalent cations; (d) pressing the suspension of broken animals through a system of regularly spaced capillary constrictions of free organs; and (e) enrichment of glands by differential sedimentation and removal of contaminating material under a dissecting microscope. The nuclear isolation procedure is a large scale modification of a method previously described by Robert, using digitonin as a non-ionic detergent to solubilize cytoplasma and secretion without affecting the nuclear membrane. Nuclei obtained by this method show structural integrity and an unchanged chromosomal banding pattern. Their incorporation of UTP is within the same range as reported by other authors for nuclei by hand dissection.
在本文中，我们描述了一种从摇蚊唾液腺快速大量分离多线核的方法。腺体分离程序包括5个主要步骤：(a) 将摇蚊幼虫在液态丙烷中冷冻；(b) 在预冷的研钵中敲碎冷冻的动物；(c) 在不含二价阳离子的蔗糖培养基中解冻碎片；(d) 将破碎动物的悬浮液通过由游离器官规则间隔的毛细管缩窄系统挤压；(e) 通过差速沉降富集腺体，并在解剖显微镜下去除污染物。核分离程序是对Robert先前描述的方法的大规模改进，使用洋地黄皂苷作为非离子去污剂来溶解细胞质和分泌物而不影响核膜。通过这种方法获得的核显示出结构完整性和不变的染色体带型。它们对UTP的掺入与其他作者报道的手工解剖获得的核在相同范围内。
