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Geraint J. Parfitt, Yilu Xie, Korey M. Reid, Donald J. Brown, James V. Jester; Three-Dimensional Immunohistochemical Multiplexed High-Resolution Macroscopy (IM-HRMac) of the Meibomian Gland. Invest. Ophthalmol. Vis. Sci. 2012;53(14):592.
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© ARVO (1962-2015); The Authors (2016-present)
Iterative antibody labeling of large, 3-D volumes has not been possible using conventional immunofluorescence approaches. The purpose of this study was to develop a 3-D immunofluorescent multiplexing method coupled with high-resolution macroscopy for identification, localisation and quantification of specific cell populations in the meibomian gland.
Lower lids from a 2 year old mouse were excised for fixation in 2% paraformaldehyde. After dehydration, samples were embedded in Butyl Methyl Methacrylate and polymerised under UV light at 4oC. Once polymerised, the lids were serial sectioned for 312µm (2µm thick) and scanned using non-linear optical (NLO) imaging of second harmonic signals to identify extracellular matrix for segmentation of the glands. After NLO imaging, sections were de-plasticised, immuno-stained with anti-Ki67 (Abcam) and imaged. Antibodies were then eluted with 0.04% SDS for subsequent immuno-labeling with anti-cytokeratin 1 (CK1, Abcam). Imaging was performed using a Zeiss LSM 510 microscope. Finally, reconstructions were generated using Amira, while Fiji was used for quantification of Ki67 labeling index.
Consecutive antibody staining was successfully achieved on a meibomian gland excised from a 2 year old mouse. Ki67 was used to localise and quantify proliferating meibocytes, whilst CK1 labeling identified regions of ductal keratinisation. Ki67 positive nuclei accounted for 9.7% of the total cellular content in the whole meibomian gland, indicating a total of 801 cells actively cycling. In the duct, Ki67 labeling was generally limited to the basal cells on the ventral aspect underlying the Orbicularis muscle, whereas acini exhibited different extents of basal cell cycling. CK1 labeling was abundant on the entire lid margin and also extended into the ductal epithelium at the orifice.
In conclusion, our novel method allows mapping of multiple antigens in the meibomian gland to quantify and localise specific cell types. Stripping and re-staining of the sections was achieved four times, although unlimited re-probing is theoretically possible. In the future, this technique will be applied to young mice and dry eye models for comparative analysis to offer insights into the age-related changes that occur in the meibomian gland.
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