Eg1150

Histological analysis of the jejunum was performed to assess the effects of supplements on gut histomorphometry. Tissues were processed (Leica^®TP1020 Semi-enclosed Bench-top Tissue Processor, Nussloch, Germany), paraffin-embedded (Leica^®EG1150 Tissue Embedding Centre, Nussloch, Germany), and 4 μm sections were prepared (Leica^®RM2125 microtome, Nussloch, Germany). AB (pH 2.5)-PAS staining were carried out under standard protocol to stain both acidic and neutral goblet cells. The sections were observed under light microscope (Leica^®3000 LED, Wetzlar, Germany), and photographed (Leica DFC450 C, Wetzlar, Germany) at 200× magnification. The images were analyzed using ImageJ 1.5v software. AB-PAS positive goblet cell count, and density, as well as villi height were determined [46 (link)].

The samples were fixed with 4% paraformaldehyde (PFA) containing 2% sucrose in PBS at 4 °C overnight and embedded into paraffin using a tissue processor (EG1150, Leica, Germany). FFPE sections (3 μm thick) were cut with a rotation microtome (RM2255, Leica, Germany). Dewaxed paraffin sections were rehydrated by alcohol series, treated with 3% H₂O₂ for 10 min at room temperature and steam-heated for 2.5 min to retrieve the antigen using ethylene diamine tetra acetic acid buffer (PH = 8.0). Subsequent immunostaining was performed with a 50-min incubation period in 37 °C with the monoclonal antibodies for PD-1 and PD-L1 (Supplementary Table S2). The IHC staining of PD-1 and PD-L1 proteins was performed on two different slides. Tonsil tissue was taken as a positive control. Immunoreaction was visualized using a Peroxidase/DAB kit (Cat. K5007, Dako, Denmark). Images were taken with a phase contrast microscope (Eclipse 80i, Nikon, Japan). Details of all reagents with reference to the immunohistochemical staining procedure are listed in Supplementary Table S3.

Hypocotyl sections were fixed in FAA (50% ethanol, 5% glacial acetic acid, and 3.7% formaldehyde mixture) overnight at 4 °C. After dehydration in different concentrations of alcohol (15, 30, 50, 75, and 100%), the sample was embedded in wax. A Leica EG1150 was used to cut 10-μm-thick sections, which were stained with 0.5% toluidine blue until the tissue outline was clearly visible. All stained sections were used in hypocotyl AR primordium detection under a stereomicroscope (Leica M205 FA).

After treatment, all mice were injected with 5-Bromo-2′-deoxyuridine at a concentration of 50 mg/kg (BrdU, Sigma-Aldrich, St. Louis, MO, USA) by intraperitoneal injections administered twice a day for 4 days [69 (link)]. All mice were then euthanized using chloral hydrate (0.25 mg/mL) and the brains were dissected and fixed in 4% paraformaldehyde (4% PFA) for 48 h at 4 °C. After fixation, some of the samples were dehydrated and embedded in OCT, and kept at −20 °C until further analysis. Other samples were put into 75% alcohol, dehydrated and embedded with paraffin (Leica, EG1150, Wetzlar, Germany), and kept at 4 °C until further analysis.

After the rats were anesthetized with pentobarbital sodium (50 mg/kg bw), the eyeballs were obtained and immersed in FAS eye fixative solution for 24 h. The eyeballs were then immersed in 75%, 85%, 90%, and 95% alcohol, anhydrous ethanol, xylene, and melting paraffin for dehydration and wax dipping. Wax-soaked eyeballs were embedded in the paraffin processor (EG1150, Leica, wetzlar, Germany). The cooled wax blocks were sliced to a thickness of 4 μm on a microtome (RM2235, Leica, wetzlar, Germany). The slices were dewaxed with xylene, rehydrated in graded alcohol solutions including anhydrous ethanol, 95%, 85%, and 75% alcohol, then put into distilled water, and stained with hematoxylin solution for 5 min and eosin dye for 5 min. The tissues were then dehydrated in alcohol and xylene, sealed with neutral gum, and observed under a microscope (DM3000, Leica, wetzlar, Germany). The thickness of the retina (ILM + NFL + GCL, IPL, INL, OPL and ONL) was quantified using ImageJ software (the quantification point was 1 mm from the optic disc). The ILM and NFL are relatively thin and difficult to be accurately distinguished from the GCL on the HE-staining section, we actually included the ILM and NFL when quantifying the GCL. The average value was calculated according to 3 left eyes from 3 animals.

The samples were fixed by 4 % paraformaldehyde containing 2 % sucrose in PBS at 4 °C for overnight and embedded into paraffin using a tissue processor (EG1150, Leica, Germany). FFPE sections (3 μm thick) were cut with a rotation microtome (RM2255, Leica, Germany).