Apr 21, 2023

Public workspaceImmunocytochemistry of cultured human Medium Spiny Neurons (MSNs) V.1

DOI
dx.doi.org/10.17504/protocols.io.rm7vzb1p2vx1/v1
  • 1Oxford Parkinson's Disease Centre and Department of Physiology, Anatomy and Genetics, University of Oxford, South Park Road, Oxford OX1 3QU, United Kingdom;
  • 2Kavli Institute for Neuroscience Discovery, University of Oxford, Dorothy Crowfoot Hodgkin Building, South Park Road, Oxford OX1 3QU, United Kingdom;
  • 3Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
Cláudia C. Mendes
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzb1p2vx1/v1
Protocol Citation: Quyen Do, Richard Wade-Martins 2023. Immunocytochemistry of cultured human Medium Spiny Neurons (MSNs). protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzb1p2vx1/v1Version created by Cláudia C. Mendes
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: February 21, 2023
Last Modified: April 21, 2023
Protocol Integer ID: 77416
Keywords: Immunocytochemistry, Cell Culture, Human Neurons
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-020370
Abstract
This protocol describes the immunolabelling of one or several protein targets on PFA-fixed cell culture on glass coverslips.
Guidelines
Adherent cells are prone to peeling, and hence, addition and removal of any liquid should be performed slowly with care.
At no point during the entire procedure prior to mounting should the sample be left to dry.

Fluorescent-dye conjugated Secondary Antibodies are light-sensitive and hence should always be protected from light.

All PBS washes should be at least 10 mins, and plates are left to incubate on the bench at room temperature.
Materials
Reagents:

Equipment:
Preparing Striatal neuronal induction base medium (sNIM):
  • DMEM/F12 basal medium
  • 1% MEM Non-Essential Amino Acids (NEAA)
  • 1% Glutamax
  • 1x B27 without vitamin A
  • 1% penicillin/Streptomycin (P/S)
  • 0.05% β-mercaptoethanol

Preparing Day 16 (D16) media:
1. Add to sMM1 base media:
  • 100 ng/ml BDNF (1:10000)
  • 200 μM Ascorbic acid (1:1000)
  • 10 μM DAPT (1:10000)
  • 2 μM PD0332991 (1:500)
  • 0.6 mM CaCl2 (1:1000)
  • 1 μM LM22A4 (1:1000)
  • 200 nM cAMP (1:500)
  • 3 μM CHIR (1:3333)
  • 300 μM GABA (1:1000)
  • 25 ng/ml Activin A (1:1000)
  • 10 μM ROCKi (1:1000)
Replating human Medium Spiny Neurons (MSNs) onto glass coverslips
Replating human Medium Spiny Neurons (MSNs) onto glass coverslips
Prepare glass coverslips and plates for replating
Add 10 or 100 μg/mL of Poly-D-lysine onto plastic wells and glass coverslips, respectively and incubate at 37⁰C overnight.
Note
These glass coverslips should have been sterilised in 70% ethanol for at least 1 hour and air dried completely in a tissue culture hood.

Wash plenty with Phosphate-buffered Saline (PBS), at least 3 times.
Add Matrigel and incubate at 37⁰C for at least 1 hour.
Prepare media for thawing and replating
Pre-warm spinning falcons containing 9 mL of Neurobasal.
Prepare Day 16 (D16) media + ROCKi (1:1000).
Allow an aliquot of desired volume to reach room temperature.
Thawing and replating of Day 16 Medium Spiny Neurons (MSNs) precursors
Thaw cryovial containing Day 16 MSN precursors in water bath until only a small component remains frozen.
Carefully transfer contents of cryovial to pre-warmed spinning tubes.
Centrifuge at 350g for 5 min.
While spinning, aspirate Matrigel and replace with D16 media + ROCKi (1:1000).
Aspirate media from cell pellet in spinning falcon and replace with D16 media + ROCKi (1:1000), slowly and gently resuspending the pellet.
Transfer an appropriate amount of cell suspension into previously prepared glass coverslips and swirl plate gently in a figure 8 motion.
Allow cells to be cultured on glass coverslips as described in step 5 of Protocol: Differentiation of human medium spiny neurons (MSNs) from induced pluripotent stem cells (iPSCs) until relevant experimental timepoints for immunocytochemistry.
PFA Fixation
PFA Fixation
Transfer glass coverslips cultured with MSNs matured to relevant experimental timepoints into a 6-well plate.
Draw a fitting circle enclosing the entire coverslip with a hydrophobic Pap pen.

Note
This circle serves as a boundary to contain the fluid volume applied onto the cell coverslip.

Note
If performing immunocytochemistry on plastic well-cultured MSNs, omit steps 4 and 5 and proceed to step 6 onwards.

Fix cells by adding 2% PFA in PBS to each well at room temperature for 20 mins.
Remove PFA completely using a pipette.
Wash thoroughly 3 times with PBS, at least 10 mins each.
Incubate the coverslips in PBS for at least 1 hour at room temperature or at 4°C overnight.
Heat-induced antigen retrieval
Heat-induced antigen retrieval
Perform antigen retrieval by adding 50 mL of 1x citrate buffer (pH 6.0) to each well, and place the 6-well plate in a water bath at 80ºC for 5 mins.

Note
Fluid can quickly dried out during the incubation - a generous amount of citrate buffer should be added.

This step is time- and temperature-sensitive.

Longer incubation and/or higher temperature can damage the cell sample.

Leave to incubate at room temperature for 10-20 mins.
Prepare blocking solution containing PBS with 10% Donkey Bovine Serum (NBS) and 0.1% Triton-X during the post-antigen retrieval incubation.
Remove the citrate buffer and add 50 mL of blocking solution.
Leave to incubate at room temperature for 10 mins.
Wash twice with PBS, at least 10 mins each.
Primary Antibody Incubation
Primary Antibody Incubation
During the previous washes, prepare the Primary Antibody Solution containing PBS with 10% Donkey Bovine Serum (NBS), supplemented with appropriate primary antibodies in their respective working concentrations.

The following primary antibodies (working concentration 1:250) were used in Do, Q. et al. (2023) for immunostaining: anti-CTIP2, anti-DARPP32, anti-DARPP32, GAD67, anti-MAP2, anti-Calbindin, PDYN, PKEN, Substance P, Tyrosine hydroxylase.
Remove all the PBS from each well and add 80 mL of the Primary Antibody Solution.
Leave to incubate overnight at 4°C.
Secondary Antibody Incubation
Secondary Antibody Incubation
Remove all the Primary Antibody Solution and add fresh PBS.
Wash thoroughly 3 times with PBS, at least 10 mins each.
During the last wash, prepare Secondary Antibody Solution containing PBS with 10% Donkey Bovine Serum (NBS), supplemented with appropriate secondary antibodies and DAPI (4',6-Diamidino-2-Phenylindole, Dilactate) in their respective working concentrations.

The following secondary antibodies (working concentration 1:1000) were used in Do, Q. et al. (2023) for immunostaining: Alexa-Fluor 555 Mouse and Rabbit, Alexa-Fluor 648 Rabbit, Alexa Fluor 488 Rat and Chicken, and DAPI.

Note
Keep pre-made solution wrapped in aluminium foil and/or keep away from direct light during incubation.

Remove all the PBS from each well and add 80 mL of the Secondary Antibody Solution.
Leave to incubate at room temperature for 1-2 hours.

Note
Keep cell plates wrapped in aluminium foil and/or keep away from direct light during incubation.

Once done, wash well with PBS.
Mounting
Mounting
Wrap culture plates with aluminium foil and/or keep away from direct light.
Keep immunostained well plates in PBS plus 0.02% Azide for long-term storage.

Note
Samples can be mounted immediately after washes (step 22) without PBS storage, if imaging is to be done within the next 48-72 hours.

Keep immunostained coverslips in PBS till 24 hours prior to imaging date.

Note
Regularly add extra PBS to protect samples from drying out.

At least 24 hours prior to the recording, apply a small drop of SlowFade Diamond Antifade mountant onto each coverslip, and carefully place the coverslip on the glass slides.
Leave to incubate the mounted slides at 4°C overnight.

Note
Proceed with imaging within 2 weeks after completing the protocol.

SlowFade Diamond Antifade mountant is non-hard setting mountant, and therefore, prolonged storage of the mounted sample is not desirable.