Method Article

Quantifying the Distribution of a Presynaptic Protein in the Mouse Brain Using Immunofluorescence

July 8th, 2025

In This Article

Abstract

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Source: Wallrafen, R., et al. Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence. J. Vis. Exp. (2019).

This video demonstrates the immunofluorescence staining of a mouse brain slice to quantify the distribution of a specific presynaptic protein. The process involves blocking nonspecific sites, applying primary antibodies for the target protein and a reference marker, followed by secondary antibody labeling and nuclear counterstaining. The reference marker ensures accurate quantification by providing a consistent baseline, and the distribution of the presynaptic protein is analyzed across brain regions using confocal microscopy.

Protocol

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

1. Immunofluorescence

  1. Prepare solutions including the blocking buffer, antibody buffer, washing buffer 1, and washing buffer 2 (see Table 1).
  2. Rinse slices once with phosphate buffer (PB) to remove excess optimal cutting temperature (OCT) compound.
    1. Remove the solution with a plastic pipette without sucking in the brain slices. Add 250 µL of fresh PB with a 1000 µL pipette.
      CAUTION: Slices should not dry out, so remove ....

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Disclosures

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No conflicts of interest declared.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1.5 mL reaction tubesEppendorf30120094
50 mL reaction tubesGreiner Bio-One227261
multiwell 24 wellFisher Scientific087721H
plastic pipette (disposable)Sarstedt8,61,176
1000 mL pipetteRainin17014382
2 ml pipetteEppendorf3123000012
Vortex Genius 3IKA3340001
Menzel microscope slidesFisher Scientific10144633CF
StereoscopeLeica
LSM800ZeissConfocal microscope
PBS (10X)Roche11666789001
Tissue TekSakura4583OCT
Na2HPO4BioFroxx5155KG001
NaH2PO4Merck1,06,34,60,500
normal goat serumMerck MilliporeS26-100ML
normal donkey serumMerckS30-100ML
Triton X-100Merck1,08,60,31,000
rabbit anti-MoverSynaptic SystemsRRID: AB_10804285
guinea pig anti-SynaptophysinSynaptic SystemsRRID: AB_1210382
donkey anti-rabbit AF647Jackson ImmunoResearchRRID: AB_2492288
goat anti-mouse AF488Jackson ImmunoResearchRRID: AB_2337438
Mowiol4-88Calbiochem475904
ZEN2 blue softwareZeissMicroscopy software
FIJIImageJAnalysis software
Microsoft ExcelMicrosoft

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Tags

Presynaptic ProteinImmunofluorescence StainingMouse BrainConfocal MicroscopyFluorescence IntensityPrimary AntibodiesSecondary AntibodiesNuclear CounterstainingBrain Slice PreparationImage Analysis Fiji

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