بروتوكول الأمثل لإعداد Wholemount الشبكية للتصوير والمناعية

Summary

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Abstract

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.

Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

Introduction

Performing immunohistochemistry in delicate intact tissues runs the risk of damage during handling and transfer. This can occur either in brain slices or other thin tissue, such as degenerated retina. Additionally, there are certain methods of tissue fixation that can be advantageous for immunostaining of neuronal structures, but result in compromised structural stability, precluding their use. A particular example of this is carbodiimide-based fixation, which is superior for staining receptors and hormones^1-7 but is commonly avoided due to the instability of the fixed tissue.

Here, we describe a procedure which utilizes a hydrophilized PTFE membrane to structurally support delicate tissue, either fixed or unfixed, for a variety of staining techniques. The supporting membrane allows for the manipulation of delicate tissue before and after fixation, allowing for several steps of processing while minimizing the risk of tissue damage. Overall, this simple method to preserve tissue integrity provides the opportunity to use techniques that might otherwise be avoided. As such, this approach could also be successfully used for preparation of wide variety of tissues such as brain slices that become fragile following slicing procedures.

Protocol

In all experimental procedures, animals were treated according to the regulations in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and in compliance with protocols approved by Weill Cornell Medical College. Animals were euthanized by carbon dioxide and subsequent cervical dislocation. 1. Dissection of Wholemount Retina After the mouse is euthanized, enucleate its eyes and place them into oxygenated HEPES-buffered Ringer's solution (Table 1<…

Representative Results

Demonstrated here are two representative experiments that benefit from the use of a hydrophilized PTFE membrane to support wholemount retina during staining procedures. The first experiment, illustrates the method for fast and simple characterization of retinal vasculature, an elaborate network of blood vessels that span across multiple retinal layers (Figure 2). This approach combines bolus loading of isolectin with immersion labeling with SRH. This is an efficient method of labeling living tissue that …

Discussion

The hydrophilized PTFE membrane's high biocompatibility and transparency in solutions is advantageous when working with living tissue. This preparation has been successfully used in earlier work for patch-clamp recordings of light responses^8-10. Here, we show how this approach can be adopted for structural analysis of the wholemount retina.

The bolus loading technique can be effectively used for labeling living tissue. This technique is aided by the structural support of the mem…

Materials

Millicell Cell Culture Insert, 12 mm, hydrophilic PTFE (Biopore), 0.4 μm	Millipore	PICM01250
Insulin syringe, 1 ml	Beckton Dickinson	309659
Scissors	Fine Science Tools	15003-08	dissection
Forceps, Dumont #55, inox	Fine Science Tools	11255-20	dissection
Cryostat	Leica	various
Confocal Microscope System	Nikon	various
Capillary glass	World Precision Instruments	1B150F-4
P-97 Flaming/Brown Micropipette Puller	Sutter Instrument Co.
Picospritzer III	Parker Hannifin
Glass Bottom Culture Dishes	MatTek Corporation	P35G-0-14-C
Petri dish	Falcon	1008
Disposable Graduated Transfer Pipettes	VWR	16001-180
Multiwell plates, 24-well	Beckton Dickinson	351147
Cover glass, #1	Electron Microscopy Sciences	72200-30
Polysine adhesion slides	Electron Microscopy Sciences	63412-01
Microscope slides	Globe	1301
Liquid Blocker	Electron Microscopy Sciences	71312
VECTASHIELD mounting medium	Vector Laboratories	H-1000
OCT medium	Sakura	4583
Parafilm laboratory film	Fisher	13-374-10
Dow Corning high vacuum grease	Sigma-Aldrich	Z273554
Mouse anti-PSD95	Millipore	MABN68	antibody
Donkey anti-mouse Alexa 568	Invitrogen	A10037	antibody
Isolectin Alexa 488	Invitrogen	I21411
Chemiblocker	Chemicon	2170S
Triton X-100	Sigma-Aldrich	T9284
Sulforhodamine	Sigma-Aldrich	341738
Carbodiimide	Thermo Scientific	22980	EDC
Paraformaldehyde	Sigma-Aldrich	P6148