Mouse pituitaries were fixed in 4% formaldehyde in PBS overnight at 4 °C. The tissue was washed three times in PBS and put in 10% EDTA for 3 h. They were then dehydrated by putting them in 25%, then 50%, and then 70% ethanol for 1 h each. The pituitaries were embedded in paraffin with 4-h cycles using a Tissue Tek VIP Paraffin tissue processing machine (Miles Scientific). The embedded pituitary was cut into coronal, six-micron sections, and was analyzed by immunohistochemical markers as previously described [22, 92]. Anti-YFP antibody (1:100) was from Abcam ab6556, Batch GR3216572-1, RRID=AB_305564, and anti-Tshb (1:1000) was from the National Hormone and Peptide Program, Batch AFP967793.

Antibodies were detected using either the tyramide signal amplification (TSA) (33002 CFF488A Streptavidin HRP, Biotium, Fremont, CA) and streptavidin-conjugated Alexa-fluor 488 (1:200, S11223, Invitrogen). DAPI (1:200) was incubated on the slides for 5 min to stain nuclei. DABCO-containing permount was used to mount the slides, which were then imaged using a Leica DMRB fluorescent microscope. To quantify gene expression, a set of 8–12 slides with pituitary sections were analyzed from each founder mouse. Total TSH positive cells/set were quantified, as well as total YFP-positive, and total double-positive cells expressing TSH and YFP. A total of 270–872 thyrotropes were counted for two founders.

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