Fsc 22 frozen section media

The mice were anesthetized with an overdose of isoflurane and perfused ice-cold 0.1 M phosphate-buffered saline (PBS) into the left cardiac ventricle before the removal of their brains. The left hemisphere was dissected to collect the SN and striatum, frozen in liquid nitrogen, and stored at –80 °C until further processing. The right hemisphere was fixed in 4% paraformaldehyde at 4 °C for 48 h. The brain samples were then dehydrated in serial graded sucrose solutions (10%, 20%, 30%, and 35%, in 0.1 M phosphate buffer, pH 7.4) for a week in total and then embedded into FSC 22 frozen section media (3801480, Leica Biosystems, Nussloch, Germany). The brains were sequentially sliced into 30-micrometer coronal sections for immunohistochemistry and stored in cryoprotectant at –20 °C until they were used.

The morphology of the extrudates and the samples from dead-stop trials was analysed by cutting the frozen samples laterally to the flow direction (side view) with a cryo-microtome from Leica Biosystems GmbH (Nussloch, Germany). The cooling room temperature of this instrument was set to −14 °C, while the temperature of the sample holder was set to −12 °C. To prepare the extrudates for cutting, 4–5 cm long sections were cut out from the extrudate strand. These sections were attached to a sample holder with FSC 22 Frozen Section Media, a sectioning medium from Leica Biosystems GmbH (Nussloch, Germany). This was intended to ensure product stability on the sample holder while sectioning and was tested to have no influence on the microstructure of the product. After attachment, 40-µm sections were cut from the extrudate until smooth cut surfaces were achieved. Immediately after cutting the extrudate in the cryo-microtome, the sections were discarded, and photos of the cut surface were taken with a digital camera and a macro-objective (DMC-GH2, Lumix, Kadoma, CGH2, Lumix, Kadoma, Japan).

Dulbecco’s Modified Eagle Medium (DMEM), high glucose, pyruvate (11,995-065), Fatal Bovine Serum (FBS) (16,000-044), Penicillin/streptomycin (15140-122) and 2-mercaptoethanol (21985-023) were purchased from Thermo Fisher Scientific (Waltham, MA, United States). Surgipath Paraplast Paraffin Wax (39601006) and FSC 22 Frozen Section Media (3801480) were purchased from Leica Biosystems (Chicago, IL, United States). SIS3 (S0447), Phosphotase inhibitor cocktail (p5726), and Glycerol (G6279) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, United States). Tris-HCl Buffer (pH 6.8) (T8102-050) and Tween 20 (T9100-010) were purchased from GenDEPOT (Katy, TX, United States). Radioimmunoprecipitation assay (RIPA) Buffer (IBS-BR004) was purchased from iNtRON Biotechnology (Sungnam, Republic of Korea). EZ block Protease inhibitor cocktail (K272-1) was purchased from Biovision (Milpitas, CA, United States).

Neurons were labeled in a fluorescent cell-type–specific manner by crossing Sst-IRES-Cre, Emx1-Cre, Pvalb-Cre, Drd1-Cre, and Drd2-Cre lines with Rosa26^LSL-tdTomato. All experiments were performed using double heterozygotic 7-week-old male mice. Mouse brains were immersed in FSC22 Frozen Section Media (Cat# 3801480; Leica Biosystems) and frozen at −80 °C. Frozen tissues were placed at −35 °C for 2 h before micro-sectioning and sliced into 40-µm–thick coronal sections using a cryotome (Model CM-3050-S; Leica Biosystems). Fluorescent labeling was detected using Axio Scan.Z1 (Zeiss). A reliable number of cells for FACS-mediated cell sorting was obtained by pooling brain tissue from at least three different mice and dissociating it into individual cells.

After formalin fixation, tissue samples were sliced (5-µm sections), embedded in the standard manner, and stained with hematoxylin & eosin (HE). Fatty liver was graded according to the method of Kleiner et al., 2005 [20] (link), and using a low- to medium-power evaluation of standard HE-stained liver parenchyma to measure percentage parenchymal involvement by steatosis, graded as follows: grade 0, <5% steatosis; grade 1, 5–33% steatosis; grade 2, 33–66% steatosis; grade 3, >66% steatosis. To avoid sampling errors, all the biopsies were performed on the same lobe, and the semi-quantitative grades were assigned without knowledge of the sample treatment.
Frozen liver sections (10-µm) were embedded in FSC 22 Frozen Section Media (Surgipath, Leica Biosystems, Richmond Inc., IL, USA) and stained with Oil Red O to visualize hepatic lipids. Images of the red-stained lipid areas were enhanced with Adobe Photoshop CS6, and the size and number of lipid droplets were analyzed using ImagePro Plus 5.0, as previously described [21] (link), [22] (link). A minimum of 5 separate microscopic fields of view were used to measure the lipid droplet area of each section, at a magnification of 400×.

The morphology of the extrudates was analysed by cryo-imaging [10 (link)]. For this purpose, the frozen samples were cut laterally to the flow direction (side view) with a cryo-microtome (CM 3050, Leica Biosystems GmbH, Nussloch, Germany). A cooling room temperature of −14 °C and a sample holder temperature of −12 °C were set for the instrument. To prepare the extrudates for cutting, 4–5 cm long sections were cut out from the extrudate strand. These sections were attached to the sample holder with a sectioning medium from Leica Biosystems GmbH (FSC 22 Frozen Section Media, Nussloch, Germany). This ensured product stability on the sample holder during sectioning and had no influence on the product microstructure. By cutting 40-µm sections from the extrudate, smooth cut surfaces were obtained. Photos of the cut surface were taken immediately after cutting of the extrudate in the cryo-microtome with a digital camera (DMC-GH2, Lumix, Kadoma, Japan) and a 4608 × 3456 pixel resolution.