Cm1950 clinical cryostat

Samples were sectioned using a Leica CM1950 clinical cryostat (Leica, Wetzlar, Germany). Both PolyFreeze and gelatin embedded samples were transported on dry ice. Samples were adhered to 30 mm specimen object discs using PolyFreeze tissue freezing medium and placed into cryostat with chamber temperature set to -14°C. The PolyFreeze did not touch the tissue when gelatin embedding was used. Once fully solidified the specimen object discs were attached to the specimen head set to -20°C. Tissues were sectioned with Leica 818 High Profile Microtome Blades with a trim setting of 50 μm and a section setting of 20 μm. The blade sectioned the skin perpendicularly, allowing the epidermal and dermal layers to be cut simultaneously. A minimum of three sections were sectioned for each treatment group which were thaw mounted onto indium titanium oxide (ITO) (Bruker, Billerica, MA) slides with embedding media still present. No additional skin washing was used. The sample slides were then stored at -20°C until sublimation. All parts of the cryostat including the specimen object discs were cleaned with 200 proof ethanol and blades were replaced between each of the treatment groups.

Liver sections (10 μm) were cut with a Leica CM1950 Clinical Cryostat (Leica, Wetzlar, Germany), washed with PBS three times (3–5 minutes each time), fixed with 4% paraformaldehyde (PFA) in PBS for 10 minutes, and rinsed in PBS three times at room temperature. Samples were then blocked in 5% NFDM in PBST for 1 hour at room temperature. Rabbit anti-GFP (1:200, PA1-980A; ThermoScientific Pierce Products) was used as primary antibody in 5% NFDM in PBST and samples were incubated overnight at 4 °C. Sections were washed three times in PBS and incubated with the secondary antibody Alexa Fluor 594 (1:200, R37117; Thermo Scientific Pierce Products) and Hoechst 33342 (1:2000, H3570; Bioprobes) for 1 hour at room temperature. After incubation, slides were washed three times and mounted using FluorSave Reagent (Calbiochem). Individual images were acquired using the HS All-in-one Fluorescence Microscope BZ-9000E (Keyence, Osaka, Japan) with a 20x objective and the BZ-II Analyzer (Keyence, Osaka, Japan).

Caecal tissues including caecal patch samples were fixed in 4% formaldehyde solution at 4°C overnight. The next day, tissue samples were washed three times (10 min per wash) with filtered 0.1 M PBS. The caecal patch was excised from the caecal tissue using spring scissors. Caecal patch samples were subsequently placed into a 30% sucrose solution in distilled water overnight at 4°C for cryoprotection. Caecal patches were placed in a cryomold (Tissue-Tek Cryomold, Sakura, Finetek, USA) filled with optimal cutting temperature compound (Tissue-Tek, OCT compound, Sakura, Finetek, USA). Cryomolds containing caecal patch samples were then snap frozen using liquid nitrogen and tissue blocks stored at −80°C. Frozen caecal patch samples were sectioned at 6-micron thickness using a cryostat (Leica CM1950 Clinical Cryostat, Leica Biosystems Nussloch GmbH, Germany) and collected on positively charged slides (Thermo Fisher Scientific, Waltham, MA, USA Menzel-Glaser, Superfrost^R plus, New Hampshire, USA and stained for Haematoxylin & Eosin (H&E) to assess for overall cell density.