Jan 09, 2026

Public workspaceImmunofluorescence staining of cultured mouse hippocampal neurons

DOI
https://dx.doi.org/10.17504/protocols.io.x54v95x5pl3e/v1
  • Akio Mori1,
  • Robert Edwards1
  • 1University of California San Francisco
Akio Mori
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DOI: https://dx.doi.org/10.17504/protocols.io.x54v95x5pl3e/v1
Protocol Citation: Akio Mori, Robert Edwards 2026. Immunofluorescence staining of cultured mouse hippocampal neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v95x5pl3e/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: November 20, 2025
Last Modified: January 09, 2026
Protocol Integer ID: 233094
Keywords: Neuron culture, Immunofluorscence, immunofluorescence staining of cultured mouse hippocampal neuron, cultured mouse hippocampal neuron, cultured neuron, immunofluorescence, neuron
Abstract
This protocol describes the immunofluorescence staining of cultured neurons.
Materials
Reagents
  • 4% Paraformaldehyde (PFA) in 0.1 M phosphate buffer, pH 7.4
  • PBS (phosphate-buffered saline)
  • Normal goat serum (NGS) (G9023 Sigma-Aldrich)
  • Triton X-100 (BP151, Fisher Scientific)
  • Primary antibodies (user-defined)
  • Secondary antibodies (Alexa Fluor series or others)
  • ProLong Gold Antifade Mountant (P36930, Thermo Fisher Scientific)

Consumables

  • Glass coverslips
  • Culture dishes

Equipment
  • Nikon Ti microscope, CSU-W1 spinning disk confocal unit, Andor Zyla 4.2 sCMOS camera
  • 60× oil immersion objective (PlanApo, NA 1.40)
  • Micro-Manager software (v2.0-gamma)
  • ImageJ / Fiji (NIH)
Troubleshooting
Fixation
Remove culture medium and rinse coverslips briefly with PBS.
Fix neurons with 4% PFA in 0.1 M phosphate buffer for 10–15 min at room temperature.
Wash coverslips 3 times with PBS.
Permeabilization and Blocking
Incubate coverslips for 15–30 min in PBS containing: 4% NGS, 0.1% Triton X-100
Primary Antibody Incubation
Prepare primary antibodies in PBS containing: 1% NGS, 0.025% Triton X-100
Incubate coverslips overnight at 4°C in a humidified chamber.
Incubation
Overnight
Wash coverslips 3 times with PBS.
Secondary Antibody Incubation
Prepare secondary antibodies in PBS containing: 1% NGS, 0.025% Triton X-100
Incubate coverslips for 1 h at room temperature, protected from light.
Incubation
Wash coverslips 3 times with PBS, protecting from light.
Mounting
Mount coverslips on slides using ProLong Gold Antifade Mountant.

Imaging
Visualize fluorescence using a Nikon Ti + CSU-W1 spinning disk confocal system equipped with a 60× oil- immersion (NA 1.4) objective.
Imaging
Process images using ImageJ (NIH).