Lampbrush chromosome protocols

How to make preparations of Lampbrush Chromosomes

Because of their great length and delicate form , lampbrush chromosomes (LBCs) are difficult to study in sections of the oocyte nucleus or "germinal vesicle (GV)". This inherent structural problem was already recognized by Rückert, who isolated and examined intact but fixed GVs from shark oocytes (Rückert, 1892). Somewhat later Duryee (1937; 1941; 1950) made a major advance by showing that GVs could be removed from living oocytes and the nuclear envelope removed to allow direct access to the unfixed chromosomes and nucleoli. Duryee stressed the importance of Ca⁺⁺-free solutions for maintaining lifelike chromosome structure, and all subsequent protocols have been based on his crucial observation. Finally, attachment of the chromosomes and other nuclear organelles to a glass slide is essential for conventional or immunofluorescent staining and particularly for in situ hybridization. All recent protocols include a centrifugation step to assure attachment of the GV contents to the slide.

Freshly isolated and unfixed LBCs can only be examined at high magnification if the chromosomes are dissected from the GV into a chamber the bottom of which consists of a coverglass and then observed with an inverted phase contrast optical system, looking up through the bottom of the chamber. This technical approach was devised by J.G. Gall in 1954 and opened the way for all modern experimental studies of these remarkable objects.

Five protocols for making and examining preparations of LBCs are available here. "Getting Started" will introduce you to some of the very basic aspects of LBC technology. It is based on a successful undergraduate laboratory teaching exercise. The other 4 protocols explain how to work with LBCs from urodeles (mainly newts of the genera Triturus and Notophthalmus but including Ambystoma mexicanum (Axolotl)), Xenopus laevis, and birds (with special regard to the chicken, Gallus).

A protocol recently contributed by the St. Petersburg laboratory describes how to use in-situ nucleic acid hybridisation (FISH) on bird lampbrush chromosomes (FISH).

Each protocol can be downloaded and printed out as a WORD document, for your convenience.

Have lots of fun!

Exciting new protocols for 2018!

Two new protocols have been recently published and are available to download here:
Saifitdinova et al. 2017
Morgan 2018

Other specialist protocols for experimental approaches to lampbrushology will be added later to this site.

Introduction

People

Publications

Protocols

Picture gallery

News