T rk stain solution

The severity of the intraocular inflammation was evaluated clinically and histologically. The degree of the anterior uveitis was clinically assessed 24 hours after the LPS injection. The severity of the intraocular inflammation was graded from 0 (no disease) to 4 (severe disease), as described previously 11 (link). All eyes were examined with a binocular microscope and then scored clinically on the base of vascular engorgement, pupillary signs, and haziness of anterior chamber.
For infiltrating cell counting, the aqueous humor sample was suspended in an equal amount of Türk stain solution (Merck, Germany), and the cells were counted with a hemocytometer under a light microscope. The number of cells per field (equivalent of 0.1 μl) was manually counted, and the results of four fields from each sample were averaged to calculate the number of cells per microliter.

Twenty-four hours after LPS injection, the rats were euthanatized, and the aqueous humor was collected immediately from both eyes via an anterior chamber puncture (25–50 μl/rat) using a 30-gauge needle (Terumo Corporation, Tokyo, Japan), under an Olympus SZ61 microscope (Olympus, Tokyo, Japan). To count the cells, the aqueous humor samples were suspended in an equal amount of Türk stain solution (Merck, Darmstadt, Germany). The cells were counted with a hemocytometer under an Olympus CKX31 microscope (Olympus), and the number of cells was obtained by averaging the results of four fields from each sample. The total protein concentration in the aqueous humor samples was measured with a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). The aqueous humor samples were stored in ice water until testing, and cell counts and total protein concentrations were measured on the day of sample collection.
TNF-α levels in the aqueous humor were assessed with a commercially available rat TNF-α enzyme-linked immunosorbent assay kit according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA). Each assay was performed in triplicate.