For histology, cell cultures maintained on electrospun membrane were fixed with 4% paraformaldehyde in PBS. Samples were embedded in Cryomatrix resin (Thermo Fisher) and cryo-sectioned using a Cryostar NX50 cryostat (Thermo Scientific) to obtain 14 μm thick sections. The samples were mounted on microscopic slides (Thermo Scientific) in Prolong mounting medium with NucBlue counterstain (Thermo Fisher) and imaged with phase contrast and epifluorescence microscopy. For semi-thin sections cell cultures were fixed in 4% paraformaldehyde/1% glutaraldehyde. Samples were dehydrated in a graded series of acetone, and embedded in Spurr’s resin (Sigma). Semi-thin sections were cut with a Reichert Ultracut microtome, mounted on microscopic slides (Thermo Scientific), stained with toluidine blue and imaged with brightfield microscopy.
Microscopic slides
Manufactured by Thermo Fisher Scientific
Sourced in Germany
Microscopic slides are thin, flat pieces of glass or plastic used in various scientific and medical applications, such as microscopy and specimen analysis. They provide a transparent surface to mount and examine samples under a microscope. Microscopic slides are designed to hold and support the specimen while allowing light to pass through, enabling clear observation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Cryostar nx50 cryostat, by Thermo Fisher Scientific (1 mentions)
Cryomatrix resin, by Thermo Fisher Scientific (1 mentions)
Spurr s resin, by Merck Group (1 mentions)
Roticlear, by Carl Roth (1 mentions)
Paraformaldehyde pfa, by Merck Group (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Low melting agarose, by Thermo Fisher Scientific (1 mentions)
Prolong diamond, by Thermo Fisher Scientific (1 mentions)
Axiocam mrm ccd camera, by Zeiss (1 mentions)
Prolong glass antifade mountant with nucblue stain, by Thermo Fisher Scientific (1 mentions)
Ab2480, by Abcam (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
5 protocols using microscopic slides
1
Histological Analysis of Cell Cultures
2
Paraffin Embedding of Fixed Eyes
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Fixed eyes were incubated overnight in 70% ethanol at 4 °C prior to dehydration in a graded ethanol series. Samples were then cleared with xylene (RotiClear; CarlRoth, Karlsruhe, Germany), followed by incubation in a paraffin bath for 90 min at 64 °C. Molds were used to cast paraffin blocks containing eye samples oriented with the optic nerve-cornea axis parallel to the surface. Sections of 4 µm thickness were longitudinally sectioned on a rotating microtome (Leica Jung RM 2065; Leica Microsystems, Wetzlar, Germany) and mounted on microscopic slides (Thermo Fisher Scientific, Waltham, MA, USA).
3
Endothelial Cell Activation and NF-κB Translocation
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5 × 104 HGMVECs/well were seeded on 1% gelatin (Fluka) coated round glass coverslips Ø 1 cm (VWR) and grown to confluent monolayer. Subsequently, HGMVECs were stimulated with 25 or 50 µM heme and/or Zn(II) Mesoporphyrin IX (ZnMP; Frontier scientific) for 6 h. After stimulation, cells (n = 1 for ZnMP, and n = 3 for NF-κB) were fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich) for 15 min at RT and subsequently stained with appropriate antibodies, 2 µg/ml of CD31 (mouse IgG1; Sanquin) and 4 µg/ml of monoclonal mouse α-human NF-κB p65 (F-6; Santa Cruz)) diluted in PBS-10% goat serum for 1 h at RT. Subsequently, 4 µg/ml of secondary antibody Alexa fluor goat α-mouse IgG (Thermofisher scientific) and the nuclear stain DAPI 1:1,000 diluted in PBS was added and incubated for 1 h at RT. Hereafter, the cells were washed three times with PBS. Finally, stained cells on the coverslips were mounted on microscopic slides (Thermofisher scientific). Slides were viewed with a Zeiss fluorescence microscope or by Olympus FV1000 confocal microcopy. To present the data of the multiple experiments (n = 3) performed for NF-κB translocation, a representative slide was chosen to incorporate in the figure.
4
Lung Slice 3D Cell Culture and Imaging
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PCLSs provided a 3D cell culture model to image vascular remodeling within the lung microenvironment and were adapted from previously described protocols (22 (link), 58 (link)). For mouse PLCSs, lungs were inflated in situ with 0.4 ml of 2% low-melting agarose (Thermo Fisher Scientific) in PBS. After inflation, lungs were carefully dissected out and fixed in 4% paraformaldehyde (PFA) (Electron Microscopy Sciences) overnight at 4°C. Human lung tissue was collected from anonymized donors at Hammersmith and Royal Brompton Hospitals NHS Trusts and immediately fixed in 4% PFA overnight at 4°C. Following fixation, 100–200 μm transverse sections were prepared using a Compresstome VF-300 Vibrating Microtome (Precisionary Instruments).
PCLS were permeabilized in PBS complemented with 0.5% Triton (MilliporeSigma) for 1 hour at room temperature, then blocked in animal-free blocker (2BScientific Ltd.) for 1 hour. Slices were incubated with indicated primary antibodies overnight at 4°C in 25% animal-free blocker in PBS, and where required, PCLS were incubated with secondary antibodies for 5 hours at room temperature in 25% animal-free blocker in PBS. Lung slices were mounted on microscopic slides (Thermo Fisher), immersed in ProLong Diamond (Thermo Fisher), and kept at 4°C until image acquisition.
PCLS were permeabilized in PBS complemented with 0.5% Triton (MilliporeSigma) for 1 hour at room temperature, then blocked in animal-free blocker (2BScientific Ltd.) for 1 hour. Slices were incubated with indicated primary antibodies overnight at 4°C in 25% animal-free blocker in PBS, and where required, PCLS were incubated with secondary antibodies for 5 hours at room temperature in 25% animal-free blocker in PBS. Lung slices were mounted on microscopic slides (Thermo Fisher), immersed in ProLong Diamond (Thermo Fisher), and kept at 4°C until image acquisition.
5
Immunofluorescent Labeling of Zebrafish Ectoderm
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Zebrafish ectoderm cell aggregates were fixed in 4% paraformaldehyde in PBS for 30 min and per- meabilized in 0.1% Triton X-100 in PBS for 10 min. The non-specific binding sites were blocked by incubation in a culture medium containing 10% serum for 2 h. Primary antibody against phosphorylated (pS19) myosin light chain (rabbit, polyclonal, Anti-MYL12A phospho S19, Abcam ab2480) was applied in 1/100 dilution for 2 h at room temperature and then overnight at 4 °C. Anti-rabbit secondary antibody conjugated with AlexaFluor-555 (Southern Biotech, 4030-32) was used in 1/200 dilution for 4 h at room temperature. All incubations were followed by triple washing steps in PBS for 1 h. Finally, immunolabeled aggregates were mounted on microscopic slides (Thermo Scientific) using a mounting medium (Prolong Glass Antifade Mountant with NucBlue Stain, Invitrogen, P36981) containing NucBlue counterstain to visualize cell nuclei. Fluorescent labels were imaged using a Zeiss Axio Observer Z1 microscope with Zeiss EC Plan-Neofluar 40x/0.75 or Olympus A 100x/1.3 objectives and Zeiss AxioCam MRm CCD camera. Images were processed using NIH ImageJ software.
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