Tissue tek vip

In order to ensure consistency in cellular content for all samples, histological staining and imaging was conducted prior to transcriptomic analysis. Tissue was sampled from an area approx. 2 cm from the center of the right lung in each animal and placed immediately in 10% buffered formalin (pH 7.4). After 24 h, the samples were processed using an automated processor (Tissue-TEK VIP, Sakura Finetek) and embedded in paraffin wax. Sections of 5 μm were cut using a microtome (Leitz 1512), and mounted onto glass slides. Samples were stained using a haematoxylin and eosin stain and were visualized using Image-Pro Plus software (Version 5, Media Cybernetics) [20 (link)].

Mid-colon tissues were fixed in 10% neutral-buffered formalin for 24 hr and then transferred to 70% ethanol until embedding in paraffin (Tissue Tek VIP, Sakura Finetek USA, Inc., Torrance, CA, USA). Tissues were sectioned into 7 μm-thick slices using a micro-tome (Microm HM 310, MICROM Laborgeräte GmbH, Berlin, Germany) and mounted onto glass slides (Surgipath^® R X-Tra^® R Microscope Slides, Leica Biosystems, Buffalo Grove, IL, USA). Mounted sections were stained with Harris’ hematoxylin and eosin (H&E), and sections were blindly evaluated for the presence of colitis by a board-certified veterinary pathologist at the University of Illinois (MAW).

Whole eyes were fixed in 10% NBF for 12 h at RT and then manually dissected to obtain the engrafted liver spheroids, along with the supporting iris. Explant pieces were encapsulated in HistoGel™ (EprediaTM, Thermo Fisher) and placed in CellSafe+ Biopsy Capsules (CellPath, Newtown, UK) and submerged in 70% ethanol until processed using Tissue Tek VIP (Sakura Finetek, Alphen aan den Rijn, The Netherlands). The treated explants were embedded in paraffin and cut into 4 µm sections using a microtome (Microm HM360, Thermo Fisher). The sections were used for RNAscope™ Multiplex Fluorescent V2 Assay (ACDBio Newark, US), following the company’s protocol. Images were collected in Z-stacks on a confocal microscope (TCS SP8, Leica).

Liver histology using hematoxylin and eosin was performed on paraldehyde-preserved tissue. The liver was harvested and fixed overnight in 4% paraformaldehyde. Tissues were processed (Tissue-Tek V.I.P, Sakura Finetek, Torrance, CA) and embedded in paraffin. For representative images, samples were cut (5.0 μm) and placed on adhesive coated slides (Newcomer Supply, Madison, WI), deparaffinized, rehydrated and H&E stained (Cat. No. H-3502, Vector Laboratories, Burlington, Canada). For pathology assessment of lipid deposition, samples were imaged in triplicate for representative images.

Organs were fixed in 10% neutral buffered formalin (Sigma). After 24 hours for liver, pancreas and brain, and 48 hours for pGAT, the tissues were placed in 70% ethanol prior to processing using a Tissue‐Tek VIP (Sakura Finetek, Alphen aan den Rijn, Nederland). Tissues were embedded in paraffin and cut into 4 μm sections using a microtome (Microm HM360, ThermoFisher). The sections were used for in situ hybridization using Base Scope^TM (ACDBio, Newark, CA, USA) paired double‐Z oligonucleotide probes. The IR‐B probe (BA‐Mm‐Insr‐tv2‐E11E12) targeting 2735‐2771 of NM 001330056.1 was visualized with red colour amplification. The IR‐A probe (BA‐Mm‐Insr‐tv1‐E10E11) targeting 2700‐2735 of NM 010568.3 was visualized in green. Negative (bacterial dapb) and positive (mouse ppib and polr2a) control probes were tested. Negative controls showed an almost negligible background (Figure S5B,D,E). Images were taken using a Leica SP8 equipped with a colour camera DFC7000T (Leica). Images were collected in Z‐stacks with 1.5 μm thickness. For each section, 1‐3 images were acquired using the navigator system on the LASX software, with maximum pixel resolution and using a 20× immersion objective. By using Fiji,¹⁶ each image was searched for red and green dots by eye and each dot was annotated using the Fiji cell counter plugin.

The ileal tissues were processed (Tissue-Tek VIP; Sakura Finetek, Tokyo, Japan), embedded in paraffin wax, and cut into 5-µm thick slices. Paraffin embedding, slicing, and H & E staining were performed according to the standard procedure.