We examined the trephined corneal button of BK after ALI during full-thickness corneal transplantation. After removal of the cornea by penetrating keratoplasty, we used fine forceps to peel off the corneal endothelium and Descemet's membrane from the trephined corneal button to expose the corneal stroma. Then, we placed the endothelial cell side down on the culture dish and examined it under an inverted phase-contrast microscope (ELWD 0.3; Nikon, Tokyo, Japan). After examining the corneal endothelial cells and Descemet's membranes, we embedded them in optimal cutting temperature compound. Then, we cut frozen cross sections (10 µm) on a cryostat and air-dried them for 10 minutes. We stained the cell nucleus with hematoxylin and observed it under a light microscope.
Elwd 0
Manufactured by Nikon
Sourced in Japan
The ELWD 0.3 is a long working distance objective lens designed for Nikon's laboratory equipment. It provides a working distance of 0.3 millimeters, allowing for close-up examination of samples. The lens is optimized for high-resolution imaging and offers a numerical aperture of 0.3.
Automatically generated - may contain errors
Lab products found in correlation
Stempro 34, by Thermo Fisher Scientific (1 mentions)
4 2 hydroxyethyl 1 piperazineethanesulfonic acid, by Merck Group (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Merck Group (1 mentions)
Oleic acid, by Merck Group (1 mentions)
Linoleic acid, by Merck Group (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Transwell with an 8 μm diameter pore membrane, by Corning (1 mentions)
4 protocols using elwd 0
1
Analyzing Corneal Endothelium and Descemet's Membrane
2
Monocyte Differentiation into Fibrocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Low-density BM cells were cultured in conditions that promote differentiation of monocytes to fibrocytes (Pilling et al., 2003 (link)) as described previously (Pilling et al., 2009 (link)). In brief, CD14+ monocytes purified from low-density BM cells (purity >97%) were cultured with serum-free medium (StemPro-34; Invitrogen) supplemented with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Sigma-Aldrich), 1× nonessential amino acids (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), 2 mM glutamine (Invitrogen), 100 U/ml penicillin, 100 µg/ml streptomycin, and 1× ITS-3 (containing 10 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml sodium selenite, 0.5 mg/ml albumin, 5 µg/ml oleic acid, and 5 µg/ml linoleic acid [Sigma-Aldrich]), either in flat-bottomed 96-well plates or in Lab-Tek microscope chamber slides (CC2 slides; Thermo Fisher Scientific). Culture plates and slides were maintained at 37°C in humidified air supplemented with 5% CO2.
Cells were visualized using a phase-contrast microscope (ELWD 0.3; 10/0.25 objective lens; Nikon), photographed using a digital camera (D40; Nikon), and counted as previously described (Pilling et al., 2003 (link); 2009). The number of fibrocytes generated in culture varied by patient sample but ranged from 200 to 400 cells.
Cells were visualized using a phase-contrast microscope (ELWD 0.3; 10/0.25 objective lens; Nikon), photographed using a digital camera (D40; Nikon), and counted as previously described (Pilling et al., 2003 (link); 2009). The number of fibrocytes generated in culture varied by patient sample but ranged from 200 to 400 cells.
3
Dual Staining for Apoptotic Cell Visualization
4
Invasion and Migration Assays for HUVECs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the invasion assay, the Transwell with an 8 μm diameter pore membrane (Costar, Corning, NY) was coated with 200 μl Matrigel at 200 μg/ml and incubated overnight. Twenty-thousand HUVECs with or without transfection of siRNA targeting Islet-1 were seeded into the upper chamber of the Transwell. After 24 h of incubation at 37 °C, the cells were fixed in methanol and stained with hematoxylin and eosin (H&E), and the cells that invaded through the pores to the lower surface of the filter were counted under a microscope. Three invasion chambers were used per condition. The values obtained were calculated by averaging the total number of cells from three filters. For the migration assay, a wound-healing assay was performed. HUVECs (1 × 106) were seeded on 6 cm plates coated with 10 μg/ml type I collagen. The cells were incubated for 24 h, and then the monolayer was disrupted with a cell scraper. The cells were incubated in a medium without fetal bovine serum, and photographs were taken at 0 and 48 h in a phase-contrast microscope (Nikon ELWD 0.3 ,Tokyo, Japan). The distance of the wounded region lacking cells was measured, and the results were displayed as the wound healing rate. Experiments were performed in triplicate, and four fields of each point were recorded.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!