Computational protocol: A Genetic Basis for a Postmeiotic X Versus Y Chromosome Intragenomic Conflict in the Mouse

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Protocol publication

[…] Antibody detection was performed on surface-spread testicular cells following a protocol described previously adapted from Barlow et al. . Incubation with the primary antibody (anti-SLY1 , anti-SLX/SLXL1 or anti-H3K9me3 [Upstate] diluted 1/100) was carried out over-night in a humid chamber at 37°C. DNA-FISH, then chromosome painting were performed after antibody detection as described previously . Speer DNA-FISH was carried out using mouse BACs RP23-212A20 and RP24-310N20 (CHORI). As a control for specificity (see ), SLX/SLXL1 antibody was preabsorbed with 8 mg of SLX immunogenic peptide or with 8 mg of a noncompeting peptide (SLY peptide). For the quantification of H3K9me3 signal over the PMSC, the chromocenter domain was defined using the corresponding black and white DAPI picture. Then, H3K9me3 signal outside this chromocenter domain was measured and normalized to that of H3K9me3 signal over the chromocenter for each cell (100 cells per genotype), using Metamorph and ImageJ (See ). Slides corresponding to 3 individuals per genotype were coded and randomized before the analysis; the analysis was therefore carried out blind as to genotype. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Application Microscopic phenotype analysis
Organisms Mus musculus
Diseases Mucopolysaccharidosis VII