Apr 15, 2026

Synapse staining - mouse brain sections

DOI
https://dx.doi.org/10.17504/protocols.io.3byl4wxmzvo5/v1
  • 1Duke University;
  • 2KU Leuven;
  • 3Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Sarah van Veen
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DOI: https://dx.doi.org/10.17504/protocols.io.3byl4wxmzvo5/v1
Protocol Citation: Shiyi Wang, Sarah van Veen 2026. Synapse staining - mouse brain sections. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4wxmzvo5/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 10, 2025
Last Modified: April 29, 2026
Protocol  Integer ID: 118065
Keywords: ASAPCRN, IHC, excitatory synapses, excitatory synapse, mouse brain section, mouse brain sections this protocol, using mouse brain section, brain section, mouse
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
This protocol details a method to visualize and quantify excitatory synapses using mouse brain sections.
Materials
  • 40 μm free-floating mouse brain sections
  • normal goat serum (NGS; Thermo Fisher, Cat# 01-6201) 
  • rabbit anti-PSD95 (Cat# 51-6900, RRID:AB_2533914; Thermo Fisher Scientific) primary antibody
  • guineapig anti-Bassoon (Cat# 141 318, RRID:AB_2927388; Synaptic Systems) primary antibody 
  • Alexa Fluor 488 goat anti-rabbit IgG(H+L) (Thermo Fisher, Cat# A-11008, RRID:AB_143165)
  • Alexa Fluor 647 goat anti-guineapig IgG(H+L) secondary antibodies (Thermo Fisher, Cat# A-21450, RRID:AB_2535867) 
  • DAPI (Invitrogen, Cat# D1306)
  • Mowiol (Calbiochem, Cat# 475904)
  • Fluorescence microscope (e.g. Zeiss LSM 880 microscope)

Safety warnings
Follow institutional guidelines for the disposal of biological and chemical waste.
Ethics statement
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Permeabilization of sections
Wash the brain sections in 1x TBS containing 0.2% Triton X-100 (TBST).

Note
Use 40 μm free-floating mouse brain sections containing the visual cortex.

Blocking
1h
Block non-specific binding by incubating sections in 10% normal goat serum (NGS) diluted in TBST for 01:00:00 at Room temperature .

1h
Antibody incubation
3h
Incubate sections with rabbit anti-PSD95 (1:300) and guineapig anti-Bassoon (1:500) primary antibodies diluted in blocking buffer (10% NGS in TBST) for 2 nights at 4 °C with gentle shaking.
Wash sections with TBST (3x 10 min).
Incubate sections in Alexa Fluor 488 goat anti-rabbit IgG(H+L) and Alexa Fluor 647 goat anti-guineapig IgG(H+L) secondary antibodies, diluted 1:100 in TBST, for 03:00:00 at Room temperature in the dark.

3h
Wash sections with TBST (3x 10 min).

Note
For nuclear staining, add DAPI (1:5000) to the second wash.

Mounting
2h
Mount sections onto glass slides using Mowiol.
Allow slides to cure overnight at Room temperature in the dark.

Imaging
3h
Acquire high-magnification z-stack images using a fluorescent microscope (e.g. Zeiss LSM 880):
  • Use a 63× objective plus 1.6× optical zoom.
  • Capture 3 optical sections spaced 0.34 μm apart per z-stack.
  • Ensure consistent imaging settings for all conditions.

Note
Image within 2 days for optimal results.

Analysis
3h
Identify excitatory synapses by the colocalization of pre-synaptic (Bassoon) and post-synaptic (PSD95) puncta.
Quantify the number of co-localized synaptic puncta using SynBot, an ImageJ-based software (https://github.com/Eroglu-Lab/Syn_Bot).
Protocol references
Savage JT, Ramirez JJ, Risher WC, Wang Y, Irala D, Eroglu C. SynBot is an open-source image analysis software for automated quantification of synapses. Cell Rep Methods. 2024;4(9):100861.