Whole Mount Immunostaining of Fetal and Neonatal Mouse Ovaries

Summary

This protocol describes a method for whole mount immunolabeling of developing mouse ovaries, with applications to other small tissues.

Abstract

The ovarian reserve consists of the oocytes available to a female for reproduction and are found in primordial follicles, each containing one oocyte surrounded by several granulosa cells. The oocytes start out as primordial germ cells that form outside the gonad and migrate to the developing ovary during embryonic development. Once they reach the ovary, they become oogonia and divide by mitosis, with incomplete cytokinesis, forming clusters of interconnected cells called germ cell cysts. While in cysts, the cells enter meiosis and become oocytes. In the mouse, two days before birth, the cysts begin to break apart, and each oocyte is packaged into a primordial follicle. Only some of the oocytes survive during this process, the rest undergo apoptosis. The regulation of cyst breakdown and primordial follicle formation is only partially understood, and identifying the molecules involved is crucial to understanding this process. Whole mount immunostaining of developing mouse ovaries can be used to determine the localization of potential regulatory molecules and to assess effects on primordial follicle formation due to mutation or other molecular changes using oocyte and somatic cell markers. Ovaries are harvested from fetal or neonatal mice, fixed in formaldehyde, and blocked. The ovaries are then exposed to antibodies against a protein of interest, followed by secondary antibodies conjugated to a fluorescent tag that detect the primary antibodies. Finally, the ovaries are mounted and examined by confocal microscopy. This procedure can detect multiple proteins of interest and can be applied to other small tissues, such as fetal mouse testes.

Introduction

Whole mount immunostaining combined with confocal imaging is a powerful method for analyzing protein expression and cell morphology in small tissues without sectioning¹. In this method, tissues are first harvested, fixed, and blocked, and then exposed to antibodies against proteins of interest. The antibodies are either directly conjugated to fluorophores or followed by labeling with secondary antibodies conjugated to fluorophores that recognize the primary antibodies. The tissue is then incubated with the fluorescent dye, 4',6-diamidino-2-phenylindole (DAPI) to visualize cell nuclei. This step is important to provide spatial orientation wh....

Protocol

All procedures using mice were reviewed and approved by the Syracuse University Institutional Animal Care and Use Committee (IACUC). The following protocol is for labeling neonatal mouse ovaries on post-natal days 1–5 (see Figure 1 for schematic). The reagents and the equipment used are listed in the Table of Materials.

1. Harvesting and fixing ovaries

1. Euthanize neonatal C57BL/6 mice following the National Institutes of Health’s “Guide for the Care and Use of Laboratory Animals” and IACUC-approved animal protocols. Dissect ovaries in 1x phosp....

Results

Typical results from the immunostaining method described here are shown. Images in Figure 3, Figure 4, Figure 5, and Figure 6 were taken on a spinning disk confocal microscope using a 40x oil objective, NA 1.3, with an exposure time between 100–250 ms and laser power between 0.01 and 0.1%. In Figure 3, neonatal mouse ovaries are labeled with antibodies agai.......

Discussion

This report describes a method to fluorescently label whole ovaries for proteins of interest. This procedure has been extensively used to determine the expression of proteins during primordial follicle formation and also to characterize changes in follicle formation under different conditions^4,5,6,7,8,9,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
anti Human VASA (made in goat)	R&D	AF2030
anti-DDX4/MVH (made in rabbit)	Abcam	ab13840
anti-GCNA1/TRA98 (made in rat)	Abcam	ab82527
Bovine Serum Albumin (BSA)	Fisher	BP-1600
Cover slips 22 x 22 #1.5	Warner Instruments	64-0721
DAPI (powder)	Sigma Aldrich	D-9542	prepare a 5 mg/ml solution in dH2O
Dissection scope
Donkey anti-Goat Alexa 568	Invitrogen	A11057
Donkey anti-Mouse Alexa 488	Invitrogen	A21202
Donkey anti-Rabbit Alexa 568	Invitrogen	A10042
Donkey anti-Rabbit Alexa 647	Invitrogen	A31573
Donkey anti-Rat Alexa 488	Invitrogen	A21208
Ethanol 200 proof	Pharmco	111000200
Fine Scissors - Sharp	Fine Science Tools	14060-09	for fine work removing ovary and trimming excess tissue
Forceps (Dumont Tweezer, Style 5)	Electron Microscopy Science	72705-01 (0209-5-PO)
Goat anti-Rabbit Alexa 488	Invitrogen	A11008
Microcentrifuge Tubes 1.5 mL	Thermo Scientific	3451PK
Microscope Slides	Fisherbrand	12-550-123
Nutating Mixer	LabNet	S0500
Paraformaldehyde 16%	Electron Microscopy Science	15710
Petri Dishes 100 x 15 mm	Bioplast Manufacturing	940-10774
Phosphate Buffered Saline 1X		(Fisher) S271-1;          (Sigma) P9541-500G; (Fisher) BP332-500; (Fisher) P285-500	800 mL MQ H2O               80 g NaCl                     2.0 of KCl                         14.4 g Na2HPO4          2.4 g KH2PO4          Adjust pH to 7.4 with HCl; final volume 1 L Then dilute from 10X to 1X for use
Pin holder	Fine Science Tools	26016-12	to hold straight needle bent in a slight curve
Pipette Filter Tips	OneTip	(10uL) 1121-3810;              (20 uL) 1120-1810;          (200 uL) 1120-8810;     (1000 uL XL) 1122-1830
Pipette tips	OneTIp	(10 uL) 1111-3800;          (200 uL) 1111-1800;     (1250 uL) 1112-1820
Pipettes 0.2-2, 2-20, 20-200, 100-1000	Gilson	(0.1-2 uL) F144054M;    (2-20 uL) F144056M;   (20-200 uL) F144058M;    (100-1000 uL) F144059M
Propidium iodide, 1mg/ml in dH2O	Invitrogen	P3566
RNase A powder	Sigma Aldrich	RG513-50mg	prepare a 10 mg/ml solution in 0.1 M sodium acetate (pH 5.2). Heat to 100 oC for 15 min. Cool to RT and adjust pH by adding 0.1 volume of 1 M Tris-Cl (pH 7.4)
Straight Needle			straight needle bent into a curve for severing excess tissue from the ovary
Triton X-100	Thermo Scientific	X100-100ML
Tuberculin Syringe 28G x 1/2	Tyco/Healthcare Kendall Monoject	1180128012
Vectashield	Vector Labratories	H-1900
Wagner Scissors	Fine Science Tools	14068-12	For decapitation, and larger cuts
Wide bore pipette tip 20 ul	LabCon	1026-965-008-9