An Air-Drying Method for Chromosome Preparations from Mouse Eggs

TLDR

The protocol involves washing mouse eggs, treating them with 1 % sodium citrate for 5–15 min, placing a microdrop on a grease‑free slide, adding ~0.02 ml acetic‑alcohol fixative, air‑drying, and staining with lactic‑acetic‑orcein or 2 % toluidine blue, and is applicable from diakinesis to blastocyst. The method produces excellent chromosome spreading with minimal cytoplasm, with optimal fixative drops being three for 1–8‑cell stages and five or more for blastocysts, and treatment duration is not critical but later stages benefit from longer exposure.

Abstract

The eggs, after being washed out from the genital tract, are put into hypotonic (1%) sodium citrate and left to stand at room temperature for 5–15 minutes. The duration of treatment is not very critical, but later stages, especially blastocysts, need longer treatment. A microdrop of this solution together with the eggs is placed on a grease-free slide. A few drops of acetic alcohol (3 parts of absolute ethyl alcohol, 1 part of glacial acetic acid) are immediately expelled from another pipette, whose tip is brought just over the microdrop containing eggs. The optimal number of drops of fixative has been found to be three in the case of eggs from the one cell to eight cell stage and five or even more in the case of blastocysts. These indications were calculated for drops measuring approximately 0.02 ml in volume. Air-dried preparations are stained in lactic-acetic-orcein or in 2% aqueous solution of toluidine blue. The method can be applied to all stages from diakinesis of the first meiotic division to blastocyst. It enables one to obtain excellent scattering of nuclei and metaphase plates and good spreading of chromosomes. The remnants of cytoplasm are negligible.