Low gelling agarose

For behavior and calcium imaging experiments, larvae were embedded in 2% low gelling agarose (Sigma-Aldrich). E3 medium was added after the agarose congealed. Agarose around the tail and the OV were removed for observing tail movements and application of water pulse for behavioral experiments.

E16.5 tibias and ulnas from mTmG:Col2a1-Cre:Gdf5-CreER control and mutant mice were cleared using the PACT-deCAL technique^{29 (link),30 (link)}. Shortly, decalcified samples were washed in PBS, then embedded into a hydrogel of 4% (wt/vol) acrylamide in 1x PBS with 0.25% thermal initiator 2,2’-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (Wako, cat. No. VA-044). The hydrogel was allowed to polymerize at 37 °C for 3 h. The samples were removed from the hydrogel, washed in PBS, and moved to 10% SDS with 0.01% sodium azide, shaking at 37 °C for 4 days, changing the SDS solution each day. Samples were washed four times with 1x PBST (PBS + 0.1% Triton X-100 + 0.01% sodium azide) at room temperature (RT) over the course of a day. To label nuclei, samples were submerged in 8 µg/ml DAPI in 1x PBST gently shaking overnight at RT. Samples were washed again with four changes of 1x PBST, and the refractive index (RI) of the sample was brought to 1.45 by submersion in a refractive index matching solution (RIMS) consisting of Histodenz (Sigma) and phosphate buffer, shaking gently at RT for 2-3 days. Samples were embedded in 1% low gelling agarose (Sigma) in PBS, in a glass capillary (Brand, Germany). Embedded samples were submerged in RIMS and protected from light at RT until imaging.

Fresh tissue from human fetal cortex was obtained from autopsies performed at the Robert Debré Hospital, and Necker enfants malades Hospital (Paris). Tissues came from spontaneous miscarriages or pregnancy terminations due to kidney malformations. A piece of pre-frontal cortex was collected from one hemisphere, and transported on ice from the hospital to the lab. The tissue was divided into smaller pieces and embedded 4% low-gelling agarose (Sigma) dissolved in artificial cerebrospinal fluid (ACSF). Cerebral organoids (weeks 8–12) were embedded in 3% low-gelling agarose. Gel blocks from both tissues were then sliced with a Leica VT1200S vibratome (300-µm-thick slices) in ice-cold ACSF. Slices were infected with a GFP-coding retrovirus, diluted in DMEM-F12. After 2 h of incubation, slices were washed three times with DMEM-F12 and grown on Millicell cell culture inserts (Merck) in cortical culture medium (DMEM-F12 containing B27, N₂, 10 ng ml^–1 fibroblast growth factor (FGF), 10 ng ml^–1 epidermal growth factor (EGF), 5% fetal bovine serum and 5% horse serum) for up to 5 days for human fetal brain and 48 h for cerebral organoids. The medium was changed every day. Viruses were produced from HEK-Phoenix-GP cell line, obtained from ATCC (CRL-3215).

Adult zebrafish were placed in tanks with 10 ng/µl BODIPY 493/503 (Thermo Fisher, Waltham, USA; catalog #D3922) for 30 min in the dark. Fish were washed and then placed in new tanks with fresh water for 2 hr. Fish were washed again to remove any residual BODIPY, then anesthetized and imaged as indicated above for whole adipose tissue.
Higher resolution images of zebrafish adipocytes were acquired using the Zeiss LSM 880 inverted confocal microscope using a x10 objective. Zebrafish were lightly anesthetized with 0.2% Tricaine and mounted on a glass bottom dish (MatTek, Ashland, USA; catalog #P35G-1.5–20 C) with 0.1% low gelling agarose (Sigma-Aldrich, St. Louis, USA; catalog #A9045-25G).