Mrc5 camera

Cells were plated in 35-mm glass bottom plates from MatTek Corporation (Ashland, MA). After treatment, cells were washed with PBS and fixed in 4% paraformaldehyde at room temperature for 15 min. For Z-DNA staining, a 4% paraformaldehyde solution containing 0.1% Triton-X100 in PBS was added to cells for 15 min immediately after removal of media. Cells were then washed three times with PBS. Blocking was done in 3% BSA, 0.1% Triton-X100 in PBS. Primary antibodies: Z-DNA from Abcam (Cambridge, UK, cat# ab2079) was used at 1:200 dilution. dsRNA antibody (J2) from Scicons (Hungary) was used at 1:50 dilution. AlexaFluor 488 or 594 donkey anti-mouse (Invitrogen, cat# A21206; 1:1000) and AlexaFluor 594 donkey anti-sheep (Jackson ImmunoResearch, West Groove, PA, cat# 713-585-147; 1:500) were used as secondary antibodies. Antibodies were diluted in 0.5% BSA +0.05% Triton X100 in PBS. After each antibody incubation, cells were washed three times with 0.05% Triton X100 in PBS. For DNA counterstaining, 1 µg/ml solution of Hoechst 33342 in PBS was used. Images were obtained with a Zeiss Axio Observer A1 inverted microscope with N-Achroplan 100×/1.25 oil lens, Zeiss MRC5 camera, and AxioVision Rel.4.8 software.

Stamens were sampled before anthesis and fixed in formaldehyde–acetic acid–ethanol, dehydrated in an ethanol series, cleared with xylene, and embedded in paraffin as described previously [24 (link), 25 (link)]. Paraffin-embedded tissues were cut into 10-μm-thick sections and placed on slides coated with poly-L-lysine (Sigma). Digoxigenin (DIG)-labeled antisense and sense RNA probes were transcribed from either XhoI- or XbaI-digested pEASY-ZmCPK32ISH using either T7 (antisense) or SP6 (sense) RNA polymerase (Roche), respectively. In situ hybridization was performed as described previously [25 (link)]. After the enzyme-catalyzed color reaction, an insoluble blue precipitate was observed. Slides were visualized with a Zeiss Axioscop 40 microscope and photographed with an Mrc5 camera (Zeiss).

The extracted liver was weighed and photographed. The relative weight was calculated as the ratio of the liver weight to the body weight of the rat (expressed as %).
Liver samples were fixed in 10% buffered formalin and embedded in paraffin according to the standard protocol. Paraffin sections of 5 µm were prepared and stained using a commercial kit (Biovitrum, Saint Petersburg, Russia) according to the Mallory method. Stained liver sections were examined under an Axio Scope A1 microscope (Carl Zeiss, Oberkochen, Germany) with an MRc.5 camera (Carl Zeiss, Oberkochen, Germany). Sections were examined in a blind fashion for the presence of fibrosis.
To assess the development of liver fibrosis, the measurement of the hydroxyproline content in the tissue was also performed. The method is based on the alkaline hydrolysis of tissue and subsequent quantification of free hydroxyproline (Hyp) in the hydrolysates [8 (link)]. Liver tissue (20 mg) was added to 0.5 mL of 7 M KOH and hydrolyzed at 120 °C for 40 min. The hydrolysate was neutralized with 3.5 M of H₂SO₄ and mixed with a buffered chloramine T reagent. Then the Ehrlich reaction was performed according to the standard protocol and the absorption of the solution at 550 nm was determined using a PE-5400UV spectrophotometer (“Ekrokhim” LLC, Saint Petersburg, Russia).

Lipid crystal networks of the OG, HOG, OC, and HOC networks that were processed with oleogelation in the presence of RBW were examined using a Carl Zeiss Axio Imager 2 PLM microscope equipped with a MRc 5 camera (Carl Zeiss Microscopy, Jena, Germany). Lipid microstructures of these samples were observed at room temperature after 24 h of storage at 5 °C. PLM images were obtained at 10× and 50× magnifications. Images were processed to calculate mean particle area and percent filled area (%) of lipid crystal networks within these samples in the same manner as described above in Confocal Microscopy section.

The isolated livers were fixed in 10% buffered formalin. The liver was routinely processed into paraffin blocks. On positively charged glass slides, 4–5 µm thick sections were cut. Sections were then stained with hematoxylin and eosin (H&E) for light microscopic histopathological examination of hepatic architecture, inflammation, dysplasia, and carcinogenesis. The Masson trichrome stain was used to assess tissue fibrosis. Liver histology of different groups was compared using a Zeiss Axio microscope, and photos were taken with the attached digital Mrc5 camera (Zeiss).