Cryosectioning and Immunostaining of Mouse Retina

Summary

A protocol for preparing mouse retinal cryosections and performing immunostaining on photoreceptors is described. This article enables researchers to consistently produce mouse retinal frozen sections with well-preserved morphology and high-quality immunostaining results.

Abstract

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. These methods enable detailed examinations of tissue morphologies and the localization of specific proteins within the tissue, which provide valuable insights into disease processes and mechanisms. Mice are the most widely used model for this purpose. However, because mouse eyeballs are small and mouse retinas are extremely delicate tissues, obtaining high-quality retinal sections and immunostaining images from mouse eyeballs is typically challenging. This study describes an improved protocol for cryosectioning mouse retinas and performing immunohistochemistry. An essential point of this protocol involves coating the eyeball with a layer of super glue, which prevents deformation of the eyeballs during the processes of cornea removal, lens extraction, and embedding. This step ensures the integrity of retinal morphologies is well preserved. This protocol highlights critical technical considerations and optimization strategies for consistently producing high-quality retinal sections and achieving excellent immunostaining results.

Introduction

Cryosectioning and immunohistochemistry (IHC) are indispensable techniques in biomedical research, particularly for studying complex biological structures such as the retina¹. These advanced methodologies are integral to understanding the intricate cellular composition and molecular organization of the retina. They provide researchers with the ability to investigate retinal functionality and pathology at a detailed level, offering insights that are critical for advancing knowledge in this field.

Cryosectioning plays a vital role in maintaining the morphological integrity of retinal tissue. It ensures that the delicat....

Protocol

The procedure adhered to the guidelines established by the Association for Research in Vision and Ophthalmology for the Use of Animals in Research. Approval was obtained from the Institutional Animal Care and Use Committee (IACUC) of Sichuan Provincial People's Hospital. Male C57Bl/6J mice, aged two to three months and weighing 25-30 g, were used for this protocol. A comprehensive list of the reagents and equipment utilized in this study is provided in the Table of Materials.

1. Reagent preparation

1. 1x PBS buffer
   1. Weigh 8 g of NaCl, 1.42 g of Na₂HPO₄, 0.42 g o....

Discussion

A number of factors influence the quality of tissue sections, including the composition of the fixation solution, fixation and cryoprotection time, and embedding methods⁵. When enucleating the eyeball from the mouse, it is essential to remove the extraocular muscles and other connective tissue attached to the eyeball. If not properly removed, these tissues can cause deformation of the eyeball during extraction from the eye socket, potentially leading to retinal detachment. During the fixation proc.......

Materials

Name	Company	Catalog Number	Comments
-80 °C freezer	Haier	DW-86L626
Adhesion microscope slides	CITOTEST	80312-3161
Alexa488-Goat anti-Rabbit	Proteintech	SA00006-2
C57BL/6J mouse	The Jackson Laboratory	664
Cryosection microtome	Leica	N/A
Cryostat	LEICA	N/A
DAPI	Cell Signaling Technology	4083S
Dissecting microscope	ZEISS	3943030830
Donkey serum	Solarbio	S9100
Embedding molds	Thermo Fisher Scientific	1841
Fine dissection scissors	RWD	S13001-10
Fine forceps	RWD	F11020-11
Fluoromount aqueous mounting medium	Sigma-Aldrich	F4680
Incubator	Shanghai Yuejin	N/A
KCl	Sigma-Aldrich	1049330500
KH2PO4	Sigma-Aldrich	1048771000
Kimwipes	Thermo Fisher Scientific	FIS-06666
Laser confocal microscope	ZEISS	N/A
Microscope cover Glass	CITOTEST	80340-3610
Na2HPO4	Sigma-Aldrich	1065860500
NaCl	Sigma-Aldrich	S9888
NaOH	Sigma-Aldrich	1091371003
O.C.T compound	Sakura	4583
Pap pen	Sigma-Aldrich	Z672548
PFA	Sigma-Aldrich	441244
Rabbit anti-PDE6B	Proteintech	22063-1-AP
Shaker	SCILOGEX	8042210200
Spring scissors	RWD	S11036-08
Sucrose	BioFroxx	1245GR500
Super glue	Deli	7147S
Tennis string (1.24 mm)	Gosen	TS761
Tribromoethanol	Macklin	T831042
Triton X-100	Solarbio	IT9100