Dec 16, 2024

Public workspaceImmunocytochemistry with Primary Cultured Hippocampal Neurons

DOI
dx.doi.org/10.17504/protocols.io.eq2ly6j2egx9/v1
  • Mukesh Kumar1,
  • Timothy A. Ryan1,2
  • 1Department of Biochemistry, Weill Cornell Medicine, New York, NY 10065;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, Maryland 20815, USA
Mukesh Kumar
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DOI: dx.doi.org/10.17504/protocols.io.eq2ly6j2egx9/v1
Protocol Citation: Mukesh Kumar, Timothy A. Ryan 2024. Immunocytochemistry with Primary Cultured Hippocampal Neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly6j2egx9/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 27, 2024
Last Modified: December 16, 2024
Protocol Integer ID: 114395
Keywords: ASAPCRN
Funders Acknowledgements:
NIH
Grant ID: NS036942
NIH
Grant ID: NS11739
ASAP
Grant ID: ASAP-024404
Abstract
This protocol describes a detailed method for performing immunocytochemistry (ICC) on hippocampal neurons cultured from post-natal day 0-1 pups.
Guidelines
The protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Materials
  • Hippocampal neurons: DIV-14
  • ReagentPoly-D-lysine hydrobromide (PDL)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P7886
  • 4% paraformaldehyde (ThermoScientific, J19943.K2) + 10% sucrose (SigmaAldrich, S0389)ReagentSucroseMerck MilliporeSigma (Sigma-Aldrich)Catalog #S0389
  • ReagentTriton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787
  • ReagentGoat serumMerck MilliporeSigma (Sigma-Aldrich)Catalog #G9023
  • Primary antibodies:
ReagentDDHD2 Polyclonal antibodyProteintechCatalog #25203-1-AP ,ReagentSynapsin1/2 antibodySynaptic SystemsCatalog #106 004 , ReagentMonoclonal Anti-TOMM20 antibody produced in mouseMerck MilliporeSigma (Sigma-Aldrich)Catalog #WH0009804M1 , ReagentNeuron-specific beta -III Tubulin AntibodyR&D SystemsCatalog #MAB1195 ,
NeuN (Abcam, ab104224, ab177487)ReagentAnti-NeuN antibody [1B7] - Neuronal MarkerAbcamCatalog #ab104224 ,ReagentAnti-NeuN antibody [EPR12763] - Neuronal MarkerAbcamCatalog #ab177487
GFAP (Abcam, ab7260ReagentAnti-GFAP antibodyAbcamCatalog #ab7260
ReagentGFAP AntibodyNovus BiologicalsCatalog #NB300-141 ), ReagentGM130 (E9Z6S) Rabbit mAb #70767Cell Signaling TechnologyCatalog #70767T
  • Alexa Fluor-conjugated secondary antibodies (All from Invitrogen)
  • ReagentBODIPY™ 493/503Life TechnologiesCatalog #D3922
  • MDH (Abgent, SM1000a)
  • ReagentProLong Diamond Antifade MountantThermo Fisher ScientificCatalog #P36970
  • ReagentPBS, pH 7.4Thermo FisherCatalog #10010031

Equipment

  • Dissection tools
  • Humidified chamber (Custom made)
  • Frosted microscope slides (Avantor, 16004-422)
  • Confocal Microscope (Zeiss 880)












Neuronal Culture Preparation
Neuronal Culture Preparation
Isolate hippocampal neurons from post-natal day 0-1 pups using standard dissection techniques.
Plate the neurons sparsely onto glass coverslips coated with poly-D-lysine.
Maintain neurons in culture media under standard conditions (Temperature37 °C , 5% CO₂) until they reach 14 days in vitro (DIV-14).

Temperature
Fixation
Fixation
25m
25m
Prepare a fixative solution containing 4% PFA and 10% sucrose in PBS.
Fix the neurons by gently adding the fixative solution and incubating at TemperatureRoom temperature for Duration00:10:00 .

10m
Incubation
Temperature
Rinse the fixed neurons 3 times with PBS (5 minutes each wash).
Rinse the fixed neurons with PBS for Duration00:05:00 each wash (1/3).

5m
Wash
Rinse the fixed neurons with PBS for Duration00:05:00 each wash (2/3).

5m
Wash
Rinse the fixed neurons with PBS for Duration00:05:00 each wash (3/3).

5m
Wash
Permeabilization
Permeabilization
20m
20m
Permeabilize the fixed neurons by incubating in 0.25% Triton X-100 in PBS for Duration00:05:00 at TemperatureRoom temperature .

5m
Incubation
Temperature
Wash the permeabilized neurons 3 times with PBS (5 minutes each wash).
Wash
Wash the permeabilized neurons with PBS for Duration00:05:00 each wash (1/3).

5m
Wash
Wash the permeabilized neurons with PBS for Duration00:05:00 each wash (2/3).

5m
Wash
Wash the permeabilized neurons with PBS for Duration00:05:00 each wash (3/3).

5m
Wash
Blocking
Blocking
1h 10m
1h 10m
Prepare a blocking solution containing 5% goat serum in PBS.
Incubate the neurons in the blocking solution for Duration01:00:00 at TemperatureRoom temperature .

1h
Incubation
Temperature
Gently wash the neurons once with PBS for Duration00:10:00 .

10m
Wash
Primary Antibody Incubation
Primary Antibody Incubation
10m
10m
Dilute the primary antibodies in a solution containing 5% goat serum and 0.05% Triton X-100 in PBS, following the manufacturer’s instructions.
Apply the primary antibody solution to the neurons and incubate DurationOvernight at Temperature4 °C in a humidified chamber.

10m
Overnight
Temperature
Washing
Washing
15m
15m
Wash the neurons 3 times with PBS (5 minutes each wash).
Wash
Wash the neurons with PBS for Duration00:05:00 each wash (1/3).

5m
Wash
Wash the neurons with PBS for Duration00:05:00 each wash (2/3).

5m
Wash
Wash the neurons with PBS for Duration00:05:00 each wash (3/3).

5m
Wash
Secondary Antibody Incubation
Secondary Antibody Incubation
1h
1h
Dilute the Alexa Fluor-conjugated secondary antibodies (Alexa 488, Alexa 546, Alexa 647) to a 1:500 dilution in 0.05% Triton X-100 in PBS.
Apply the secondary antibody solution to the neurons and incubate for Duration01:00:00 at TemperatureRoom temperature in a humidified chamber.

1h
Incubation
Temperature
Washing and Lipid Droplet Staining
Washing and Lipid Droplet Staining
30m
30m
Wash the neurons twice with PBS (5 minutes each wash).
Wash
Wash the neurons with PBS for Duration00:05:00 each wash (1/2).

5m
Wash
Wash the neurons with PBS for Duration00:05:00 each wash (2/2).

5m
Wash
For lipid droplet staining: Prepare a staining solution containing Amount1 µL BODIPY or Concentration100 micromolar (µM) MDH in PBS. Perform the third wash using this solution, incubating for Duration00:10:00 .
10m
Incubation
Pipetting
Wash
Gently wash the neurons with PBS for an additional Duration00:10:00 .

10m
Wash
Mounting
Mounting
Carefully mount the glass coverslips onto glass slides using Prolong antifade mounting media containing BODIPY or MDH to prevent photobleaching.
Seal the coverslip with nail-polish to prevent drying.
Imaging
Imaging
Use a confocal or fluorescence microscope to acquire images of the stained neurons.

Note
• Bovine Serum Albumin (BSA) can be used in place of Goat serum.
• Maintain gentle handling of neurons throughout the protocol to prevent damage of synapses.
• Protect fluorescently labeled samples from light to preserve signal intensity.
• Imaging conditions should be optimized to capture fluorescence signals without significant photobleaching.

Imaging