Computational protocol: ISL1-based LIM complexes control Slit2 transcription in developing cranial motor neurons

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Protocol publication

[…] To quantify ISL1 immunoreactivity in FBM neurons, 12 μm-thick transverse sections of r4 hindbrains were immunolabeled with anti-ISL1/2 and PHOX2B antibodies. The positions of Isl1-null FBM neurons were identified from the expression of PHOX2B. The background-subtracted pixel intensities of FBM nuclei were measured using MetaMorph software (Molecular Devices). To count the number of BM and SM neurons in each rhombomere, at least 3 sections from 3 embryos were analyzed for each group. The numbers of GFP, MNR2 and TBX20-expressing cells were determined in 12 μm-thick transverse sections after immunohistochemical staining. The number of nucGFP-expressing cells in the hindbrain was measured in the region medial to FBM nucleus in r2 and r4, as defined by TBX20 expression in the same sections. The level of ckSlit2 transcripts in the medial region of hindbrain and 250 × 650 pixel areas in the dorsal spinal cord were measured in adjacent transverse sections using ImageJ software. In the spinal cord, the background-subtracted pixel intensities of GFP in 250 × 650 pixel areas in the dorsal spinal cord were measured using ImageJ. At least 9 sections from 3 chick embryos were analyzed in each group. Statistical significance was analyzed by unpaired Student’s t-test or Mann-Whitney rank sum test as indicated in figure legends. To measure the length of trigeminal and FBM axons, peripheral projections from the exit point to the nerve terminals were manually traced. To calculate mean axon thickness, axon bundles were divided into 5 parts from proximal to distal and thickness at each intersection was averaged. All quantifications in images were analyzed in MetaMorph software (Molecular Devices). […]

Pipeline specifications

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