Alexa fluor 488 rat anti mouse cd45 receptor antibody

The blood and lymphatic vessels in corneal wholemounts were double stained as described previously.³³ (link)^,35 (link) Excised corneas were rinsed in PBS and then fixed in acetone for 20 minutes. After washing in PBS 3 times, and blocking with 2% bovine serum albumin (BSA) in PBS, the corneas were stained overnight (in dark, at 4°C) with rabbit anti-mouse LYVE-1 (1:200; AngioBio, Del Mar, CA, USA) for lymphatic vessels and with an FITC-conjugated rat anti-mouse CD31 antibody (1:100; BD Pharmingen, BD Biosciences, San Jose, CA) for blood vessels. On the next day, a goat-anti-rabbit Cy3-conjugated secondary antibody (1: 100; Dianova) was added to detect LYVE-1. Finally, samples were mounted on slides using fluorescence mounting media (Sigma). In indicated experiments, corneas were stained with Alexa Fluor 488 rat anti-mouse CD45 receptor antibody (BD Pharmingen, Heidelberg, Germany) to quantify CD45+ cells.
Stained wholemount images were assembled automatically from 9 to 12 images taken at × 100 magnification with a fluorescence microscope (BX53; Olympus Optical Co., Hamburg, Germany). Afterward, the areas covered with blood and lymphatic vessels (or CD45+ cells) were detected with an algorithm established in the image analyzing program Cell^F (Olympus Soft Imaging Solutions GmbH, Münster, Germany), as previously described.³³ (link)

Corneal flatmounts were prepared and double stained as previously described.33, 34, 35 Corneal BVs were detected in green with the use of rat anti‐mouse FITC‐conjugated CD31 antibody (1:100; BD Biosciences, San Diego, CA), and LVs were stained in red with the use of rabbit anti‐mouse lymphatic vessel endothelial hyaluronan receptor 1 (LYVE‐1) antibody (1:200; AngioBio, Del Mar, CA) followed by Cy3‐conjugated goat anti‐rabbit secondary antibody (1:100; Jackson ImmunoResearch [Dianova], West Grove, PA) staining. In addition, corneas were stained with Alexa Fluor 488 rat anti‐mouse CD45 receptor antibody (BD Pharmingen, Heidelberg, Germany). Cell nuclei were labeled with 4’,6‐diamidino‐2‐phenylindole (DAPI). Nine to 12 digital pictures of double‐stained flatmounts were taken automatically with the use of a fluorescence microscope (BX53; Olympus, Hamburg, Germany) at magnification ×100 and assembled into a whole image (multi‐image alignment). Thereafter, the areas covered by BVs, LVs, LYVE‐1⁺ macrophages, or CD45⁺ cells were estimated with use of the Cell^F image analyzing program (Olympus, Münster, Germany) as described previously.30