Leica dm2000 led

For general histological observations, the plant material was fixed in a solution of 60% ethanol and glycerol in a ratio of 9:1 as a softening procedure of the tissues prior to sectioning [98 ]. Temporary preparations of hand-cut transverse sections were made from the middle part of the leaf and stem of the plant. For the presence of lipids in tissues, the sections were treated with a Sudan staining solution (Sudan III in 70% ethanol) for 20 min, rinsed in 50% ethanol, and mounted in 50% glycerol [99 ]. To reveal the presence of terpenes in the tissues, the sections were treated with a NADI staining solution (0.5 mL of 0.1% α-naphthol, 0.5 mL of 1% N,N-dimethyl-p-phenylenediamine, and 49 mL of 0.1 M sodium phosphate buffer, pH 7.2) for 1 h, rinsed in 0.1 M sodium phosphate buffer, pH 7.2 for 2 min, and immediately analyzed under a microscope [100 ]. The image analysis was performed using a light microscope (Leica DM 2000 LED, Leica Microsystems, Wetzlar, Germany) equipped with a digital camera (Leica DMC 2900) and software for processing images (Leica Application Suite, LAS).

Paraformaldehyde-fixed tissues in paraffin were cut and stained with primary antibodies against corin, pro-ANP/ANP, NPR-A, β-ENaC, CFTR, SMA, and cytokeratin 18. Horseradish peroxidase (HRP)-conjugated or Alexa 488 (green)- or 594 (red)-labeled secondary antibodies were used for immunohistochemistry and immunofluorescent staining, respectively. Experimental details are described in S1 Text. Stained sections were examined with light (Leica DM2000 LED, Leica Microsystems, Wetzlar, Germany) and confocal (Olympus FV1000, Olympus, Tokyo, Japan) microscopes.

The membrane and RNAs loaded in EV were fluorescently labelled by adding five microliters of Vybrant™ DiD (Thermofisher, Belgium) and SYTO™ RNASelect™ (Thermofisher, Belgium) to one ml of isolated EV and incubated at room temperature for two hours. Free dyes were removed from labeled EV using Sepharose CL-2B (17-0140-01, VWR) SEC as described previously. Pooled EV fractions (F4 and F5) were concentrated using Amicon-Ultra 0.5 Centrifugal Filter Units with 10 kDa cutoff (UFC501096, Merck) at 4 °C.
To compare the uptake of different sized based fractions of EV, hCMEC/D3 cells were grown in pre-coated eight-well culture plates, for 24 h before EV incubation. 10⁹ labeled EV were added to cells and incubated for 24 h. After incubation, cells were washed with PBS and fixed with PFA 2%. Nuclear staining was performed with 4’,6-diamidino-2-phenylindole (DAPI) at a final concentration of 10 µg/ml for 30 min at 37 °C. Following the staining, images were captured using a Leica DM2000 LED (Leica Microsystems, Heidelberg, Germany) attached to a digital camera and Leica Application Suite X (LAS X) software (Leica Microsystems).

After BAL sample collection, lungs were removed. The left lungs were frozen in liquid nitrogen and stored at −80 °C for future studies while the right lobules were immediately stored in 4% formaldehyde for histopathological analysis. Right lobules were then fixed in 10% buffered formalin for 24 h, sectioned and processed by dehydration in ascending series of alcohol and embedding in paraffin wax. Next, 3 μm thick sections were obtained from the paraffin blocks and stained with haematoxylin and eosin (HE). The histological examination was performed by a pathologist in a blinded manner using an optical microscope (Leica DM 2000 LED, Leica Microsystems, Wetzlar, Germany). Lung injury was scored according to the following variables: centrilobular inflammation, interstitial inflammation, bronchiolitis, peribronchiolar fibrosis, presence of giant cells and interstitial fibrosis. These features were graded using 4-point scales: 0 = regular tissue, 1 = mild changes, 2 = moderate changes, 3 = significant changes. Presence or absence of giant cells and arteriolar muscularization was also recorded. Pictures were taken from representative findings at 20× and 40× magnification. An example photo of each score found from representative findings is provided in Supplementary Figure S2.

The plant material had been fixed in a solution of 50% ethanol and glycerol in a ratio of 7:3 for 24 h to soften the tissues. Hand-cut transverse and tangential longitudinal sections were made from the stem and leaf of the plant. Terpene histochemistry was examined using NADI reagent (0.5 mL of 0.1% α-naphthol (Merck KGaA, Darmstadt, Germany), 0.5 mL of 1% N,N-dimethyl-p-phenylenediamine (Merck KGaA, Darmstadt, Germany), and 49 mL of 0.1 M sodium phosphate buffer, pH 7.2 (Merck KGaA, Darmstadt, Germany) [77 ]. Cross sections were incubated with NADI reagent for 1 h in the dark, washed in sodium phosphate buffer (0.1 M, pH 7.2) for 2 min, and mounted in the same buffer. Lipid histochemistry was examined using Sudan III (Merck KGaA, Darmstadt, Germany). The sections were treated with Sudan III in 70% ethanol for 30 min, rinsed briefly in 80% ethanol, and mounted in 50% glycerol [77 ]. The sections were observed with a light microscope (Leica DM 2000 LED, Leica Microsystems, Wetzlar, Germany) equipped with a digital camera (Leica DMC 2900) and image processing software (Leica Application Suite, LAS).

Liver tissues were fixed in a 4% paraformaldehyde solution for 24 h. The fixing solution was cleared with 70% ethanol. Then, the samples were dehydrated with 70% ethanol for 40 min, 80% ethanol for 40 min, 95% ethanol for 1 h, and 100% ethanol for 1 h. After dehydrating, the samples were soaked in a mixture of xylene and ethanol (1:1) for 30 min, and then in xylene for 1 h. The processed samples were embedded in paraffin at 55 °C, followed by sectioning and baking, and 5 µm paraffin sections were obtained. Then, a part of each section was stained with hematoxylin & eosin (H&E), sealed with natural gum, and finally observed under a light microscope (Leica DM2000 LED, Leica Instruments Co., Ltd., Wetzlar, Germany) and photographed with a 3DHISTECH scanner (Jinan Tangier Electronics Co., Ltd., Jinan, China).