Microscopic Analysis of Synapses in Mouse Hippocampal Slices Using Immunofluorescence

Abstract

Source: McLeod, F., et al. Evaluation of Synapse Density in Hippocampal Rodent Brain Slices. J. Vis. Exp. (2017).

This video demonstrates a method for visualizing synaptic markers in hippocampal brain slices using confocal immunofluorescence analysis. The slices are immunostained with primary antibodies targeting presynaptic and postsynaptic markers, followed by fluorophore-conjugated secondary antibodies. The labeled markers are then visualized using confocal microscopy to evaluate the synaptic architecture through their colocalization.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Acute Hippocampal Slice Preparation

1. Remove the mouse brain as quickly as possible, using a spatula and sharp scissors to cut the skull, and place it in ice-cold, oxygenated (95% O₂, 5% CO₂), high-sucrose artificial cerebrospinal fluid (ACSF, ~100 mL).

Results

Table 1: Buffers used for immunofluorescent staining.

Blocking/permeabilizing Buffer

Concentration

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Vibratome	Leica	VT1000S
Primary antibody	Millipore	AB5905	vGlut1 (1:2000)
Primary antibody	Thermo Scientific	MA1-046	PSD-95 (1:500)
Primary antibody	Synaptic Systems	131 004	vGAT (1:500)
Primary antibody	Synaptic Systems	147011	Gephyrin (1:500)
Primary antibody	Abcam	ab5392	MAP2 (1:10000)
Secondary antibody	Jackson Laboratories	706-545-148	Donkey Anti-Guinea Pig Alexa Fluor 488 (1:600)
Secondary antibody	Abcam	ab175470	Donkey Anti-Rabbit Alexa Fluor 568 (1:600)
Mounting medium	SouthernBiotech	00-4958-02	Fluoromount-G
Confocal Microscope	Olympus	NA	FV1000 confocal microscope using a 60x 1.35 numerical aperture (NA) oil objective. For vGlut1, use a 488 laser with a percentage power of 14%. For PSD-95 use a 559 laser with a percentage power of 20%.
Analysis software	PerkinElmer	NA	Volocity
Scalpel	Swann-Morton	203	No 11
Super Glue	Loctite	1446875
95% O2, 5% CO2	BOC	NA	Cylinder
24-well plate	Corning	3524
Glass slides	Thermo	7107	08-1.0mm thick
Petri Dish	Corning	430167
iDkk1 mice	N/A	N/A	Mice were obtained by crossing tetO Dkk1 transgenic mice with CaMKIIα rtTA2 transgenic mice. TetO Dkk1 transgenic mice and CaMKIIα rtTA2 were crossed in a heterozygous state. Both mouse lines were bred in a C57BL/6J background. Single transgenic and WT littermate were used as controls.
Reagents for solutions:
NaCl	Sigma	V800372
KCl	Sigma	P9333
Na2HPO4	Sigma	255793
KH2PO4	Sigma	P9791
CaCl2	Sigma	223506
MgCl2	Sigma	M9272
NaH2PO4	Sigma	71505
Kynurenic acid	Sigma	K3375
NaHCO3	Sigma	S5761
Pyruvic acid	Sigma	107360
EDTA	Sigma	E6758
D-Glucose	Sigma	G5767
Sucrose	Sigma	S8501
Triton-X	Sigma	T8787
Donkey Serum	Millipore	S30-100ml
PFA	Sigma	P6148