Perilipin

In order to determine if the mouse ESCs have properly differentiated into brown adipocytes, immunocytochemistry analysis of UCP1 (brown adipocyte-defining marker), perilipin (surface marker of adipocytes) and PPARγ2 (adipocyte transcription factor) was performed. Mouse ESCs within the alginate microstrands were fixed using a 4% paraformaldehyde solution (Sigma-Aldrich), permeabilized using 0.2% Triton-X 100 (Sigma-Aldrich) in physiological saline, and blocked using 5% FBS in physiological saline (StemCell Technologies). Samples were incubated with the primary antibodies of perilipin (Santa Cruz Biotechnology, Inc., Dallas, Texas), PPARγ2 (Santa Cruz Biotechnology, Inc.), and brown adipocyte-defining UCP-1 (Santa Cruz Biotechnology, Inc.), at 4°C overnight or room temperature for 1 hour. The immunoreactivities were detected using secondary antibodies Alexa Fluor® 488 (perilipin) and Alexa Fluor® 647 (PPARγ2 or UCP1). Total cell population was revealed by staining cell nuclei with DAPI. All samples were imaged using the Leica Sp5 confocal microscope (Leica Microsystems, Wetzlar, Germany).

Prox1-Cre-ER^T2xRosa26^LSLtdTomato small intestine with attached mesentery was fixed in 4% paraformaldehyde + 30% sucrose overnight. The mesenteric-gut wall interface distal to the anastomosis in the portion of the ileum undergoing compensatory adaptation was then transferred into phosphate-buffered saline for fluorescent imaging using a stereoscope (Leica M205FA). The gut-mesentery interface was prepared for whole mount imaging by first staining lymphatics (Prox1, 1:1000, rabbit polycolonal, Origene, DF3516) and adipocytes (Perilipin; 1:200, goat polyclonal, Santa Cruz Biotechnology, SC-47322). Tissue was dehydrated and cleared using methyl salicylate (Sigma-Aldrich) as previous reported [30 (link)] and imaged using a confocal microscope (Leica SP8). Blinded image analysis, including lymphatic branch and valve counting of the mesentery distal to the anastomosis from the lymph node to the gut wall, was performed using Imaris (Bitplane, Switzerland) and FIJI (ImageJ) software (National Institute of Health, USA).