Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

Summary

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

Abstract

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Introduction

Retinal ganglion cells (RGCs) express L-, N-, P/Q- and T-type VGCC as determined through pharmacological blockade of these components of the whole cell Ca channel current^1,2. VGCC are transmembrane multimeric proteins that are involved in transmitter release, gene transcription, cell regulation and synaptic plasticity^3,4,5. Functional VGCCs are made up of at least three distinct classes of subunits: large, transmembrane α­₁ pore forming subunits that establish the channel’s biophysical and pharmacological properties, primarily extracellular auxiliary α­₂δ subunits and intracellular β subun....

Protocol

All experiments were carried out in accordance with the guidelines for the welfare of experimental animals issued by the U.S. Public Health Service Policy on Human Care and Use of Laboratory Animals and the University of California-Los Angeles (UCLA) Animal Research Committee. Male and female adult Sprague-Dawley rats (Charles River Lab, Wilmington, MA) between the age of 3-5 weeks were used.

1. Animal and Tissue Preparation for Wholemount Retina and Immunohistochemistry Protocol

1. Prepare the following materials and tools: A dissecting microscope, 2 forceps with very fine tips, scissors, cellulose filter paper, plastic pipette an....

Results

Immunolabeling with specific antibodies provides a platform to study the localization of particular proteins of interest in the retina and the calcium imaging technique permits the study of the contribution of the VGCCs to the calcium dynamics in the retinal ganglion cells and their axons.

By using an antibody against the ganglion cell protein RBPMS (RNA binding protein with multiple splicing) that selectively labels RGCs¹⁹, we were able to show that the Fluo-4 labeling is limited t.......

Discussion

In this article, we have described two different techniques: 1) Immunohistochemistry to show Fluo-4 localization to the ganglion cells in retinal wholemounts, and 2) Calcium imaging to analyze calcium dynamics in retinal ganglion cells and their axons.

Immunohistochemistry using wholemounts has been used to reveal the localization of proteins in the rodent retina^20-23. However, there are a few limitations. The quality of the staining relies heavily on the ability of the antibody to .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Straight scissors	WPI	14124-G	dissecting tools
Straight Forceps	WPI	501985	dissecting tools
curved iris scissors	WPI	504487	dissecting tools
Cellulose filter paper	Millipore	HABP04700
Hibernate A	Invitrogen	A12475-01	Media
Vectashield	Vector	H-1000	Mounting Media for Fluorescence
Fluo-4 pentapotassium	Invitrogen	F-14200
Isoflurane	Abbott Laboratories	05260-05	anesthesia
Microscope slide	Fisher Scientific	22-178-277
Zeiss LSM 5 Pascal microscope	Zeiss
Axioplan 40X (NA 0.8) objective lens	Zeiss
Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L) Antibody	Molecular Probes	A-11034	Secondary antibody
RBPMS	ProSci, Poway, CA		A rabbit polyclonal antibody was generated against the N-terminus of the RBPMS polypeptide (RBPMS4-24), GGKAEKENTPSEANLQEEEVR, by a commercial vendor (ProSci, Poway, CA).