Computational protocol: The Dwarf Phenotype in GH240B Mice, Haploinsufficient for the Autism Candidate Gene Neurobeachin, Is Caused by Ectopic Expression of Recombinant Human Growth Hormone

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Protocol publication

[…] This experiment was performed as described before . Briefly, naive mice were euthanized by CO2 asphyxiation followed by immediate decapitation. The neural and intermediate lobes of the pituitary were discarded using a stereomicroscope. The anterior lobe (anterior pituitary) was isolated and dissociated into individual cells with trypsin. Cells were resuspended in serum-free defined medium (SFDM; Invitrogen) and spun down on Cytospin slides (SuperfrostPlus; Thermo Scientific, Rockford, USA). Cells were permeabilized with 0.5% saponin (Sigma-Aldrich, St. Louis, USA) after fixation in 4% paraformaldehyde, and were incubated overnight with antibodies against murine GH (1/10,000), prolactin (PRL) (1/10,000), adrenocorticotropic hormone (ACTH) (1/5,000) and α-glycoprotein subunit (αGSU) (1/1,000). Hormone antisera were obtained from Dr. A. F. Parlow (National Hormone and Peptide Program, Harbor-UCLA Medical Center, Torrance, CA). Subsequently, cells were incubated for 1 h with fluorescently labeled secondary antibodies (Invitrogen; 1/1,000) and 6-diamidino-2-phenylindole (DAPI). Images were captured using an Olympus IX81 microscope. Cells were counted using ImageJ (NIH, Maryland, USA) in at least 5 random cytospin fields (1,000–2,000 cells per hormone per group) and the proportions of immunopositive cells on the total anterior pituitary cells were determined. To take into account the smaller size of the anterior pituitary and the correspondingly lower number of total anterior pituitary cells (about 2-fold, see Results) in the Nbea+/− GH240B mice, we calculated the decrease in absolute cell number of the different cell types (using their proportion and the total number of anterior pituitary cells obtained per group of mice). [...] Data are presented as mean with standard error of the mean (SEM). Significance of differences between mean values were analyzed using (where appropriate) two-tailed t test, analysis of variance (ANOVA) or repeated measures (RM)-ANOVA procedures with Tukey test post hoc comparison using Statistica version 9.0 (StatSoft, Oklahoma, USA). All statistical tests were performed with 0.05 as the α level of significance (*: P<0.05, **: P<0.01 and ***: P<0.001). […]

Pipeline specifications

Software tools ImageJ, Statistica
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens