Research Paper Volume 5, Issue 11 pp 825—834

Figure 1. From the macroscopic to the microscopic: ICT reconstruction of the young and aged mouse eyelid. (A) Dissecting microscope images of a 5-month old and (B) 2-year old mouse eyelid. Meibomian glands (black arrowheads) were identified as whitish tissue underneath transparent conjunctiva and gland dropout was observed in the older mouse eyelids (asterisk). Eyelids were embedded in BMMA and serially sectioned; the red box highlights the areas 3-D reconstructed through sequential immunostaining, as shown in C-H (C) A 5month old mouse eyelid 2μm BMMA section stained with CK1 and DAPI before antibody elution with glycine HCl and subsequent immunostaining with (D) CK5, (E) CK6 and (F) Ki67 antibodies. The resulting overlays (G and H) show the meibomian gland parallel to the conjunctiva (CJ) and embedded in the orbicularis muscle (OM). The meibomian gland duct (DU) and orifice (O) are CK5⁺/CK6⁺, whereas acinar (AC) and skin (S) epithelium are CK5⁺/CK6⁻. CK5-based reconstructions of a (I) 5month and (L) 2year old mouse eyelid visualised the hair follicle (red arrowheads) and meibomian gland epithelia (green), confirming the truncation of meibomian glands with age. In 3-D, CK1 staining at the lid margin of (J) young and (K) old lids revealed a loss of a layer of epidermal cells posterior to meibomian gland orifices in the older lid. This caused an anterior shift of the mucocutaneous junction (white arrows) to the level of the meibomian gland orifices.