Preparation and Immunofluorescence Staining of Bundles and Single Fiber Cells from the Cortex and Nucleus of the Eye Lens

Summary

This protocol describes methods to prepare peripheral, mature, and nuclear eye lens fiber cells for immunofluorescence staining to study complex cell-to-cell interdigitations and the membrane architecture.

Abstract

The lens is a transparent and ellipsoid organ in the anterior chamber of the eye that changes shape to finely focus light onto the retina to form a clear image. The bulk of this tissue comprises specialized, differentiated fiber cells that have a hexagonal cross section and extend from the anterior to the posterior poles of the lens. These long and skinny cells are tightly opposed to neighboring cells and have complex interdigitations along the length of the cell. The specialized interlocking structures are required for normal biomechanical properties of the lens and have been extensively described using electron microscopy techniques. This protocol demonstrates the first method to preserve and immunostain singular as well as bundles of mouse lens fiber cells to allow the detailed localization of proteins within these complexly shaped cells. The representative data show staining of the peripheral, differentiating, mature, and nuclear fiber cells across all regions of the lens. This method can potentially be used on fiber cells isolated from lenses of other species.

Introduction

The lens is a clear and ovoid tissue in the anterior chamber of the eye that is made up of two cell types, epithelial and fiber cells¹ (Figure 1). There is a monolayer of epithelial cells that covers the anterior hemisphere of the lens. Fiber cells are differentiated from epithelial cells and make up the bulk of the lens. The highly specialized fiber cells undergo an elongation, differentiation, and maturation programming, marked by distinct changes in cell membrane morphology from the lens periphery to the lens center^2,3,4

Protocol

Mice have been cared for based on an animal protocol approved by the Institutional Animal Care and Use Committee at Indiana University Bloomington. The mice used to generate representative data were control (wild-type) animals in the C57BL6/J background, female, and 8-12 weeks old. Both male and female mice can be used for this experiment, since the sex of the mice is very unlikely to affect the experiment's outcome.

1. Lens dissection and decapsulation

1. Euthani.......

Discussion

This protocol has demonstrated the fixation, preservation, and immunostaining methods that faithfully preserve the 3D membrane morphology of bundles or singular lens fiber cells from various depths in the lens. The stained lens fibers are compared with SEM preparations that have long been used to study lens fiber cell morphology. The results show comparable membrane structures between both preparations. EM remains the gold standard for studying cell morphology, but immunolabeling is more challenging in SEM samples for lo.......

Materials

Name	Company	Catalog Number	Comments
100% Triton X-100	FisherScientific	BP151-500
60mm plate	FisherScientific	FB0875713A
16% paraformaldehyde	Electron Microscopy Sciences	15710
10X phosphate buffered saline	ThermoFisher	70011-044
1X phosphate buffered saline	ThermoFisher	14190136
48-well plate	CytoOne	CC7672-7548
Cover slips (22 x 40 mm)	FisherScientific	12-553-467
Curved tweezers	World Precision Instruments	501981
Dissection microscope	Carl Zeiss	Stereo Discovery V8
Fine tip straight tweezers	Electron Microscopy Sciences	72707-01
Fisherbrand Superfrost Plus Microscope Slides	FisherScientific	12-550-15
LSM 800 confocal microscope with Airyscan (63X) and Zen 3.5 Software	Carl Zeiss
Nail polish
Normal donkey serum	Jackson ImmunoResearch	017-000-121
Phalloidin (rhodamine)	ThermoFisher	R415
Primary antibody
Scalpel Feather Disposable, steril, No. 11	VWR	76241-186
Secondary antibody
Straight forceps	World Precision Instruments	11252-40
Thermo Scientific Nunc MicroWell MiniTrays (dissection tray)	FisherScientific	12-565-154
Ultra-fine scissors	World Precision Instruments	501778
VECTASHIELD Antifade Mounting Medium with DAPI	Vector Laboratories	H-1200
Wheat germ agglutinin (fluorescein)	Vector Laboratories	FL-1021-5