Jan 24, 2024

Public workspaceImmunocytochemistry for the characterization of hiPSC to Motor Neuron differentiation V.3

DOI
dx.doi.org/10.17504/protocols.io.6qpvr3zbzvmk/v3
  • 1Washington University, Saint Louis. McDonnell Genome Institute (MGI);
  • 2Washington University in St. Louis
Mallory Wright
Open access
DOI: dx.doi.org/10.17504/protocols.io.6qpvr3zbzvmk/v3
External link: http://buchserlab.wustl.edu/people/
Protocol Citation: Mallory Wright, Colin Kremitzki, William J Buchser 2024. Immunocytochemistry for the characterization of hiPSC to Motor Neuron differentiation. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr3zbzvmk/v3Version created by Mallory Wright
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 24, 2024
Last Modified: March 15, 2024
Protocol Integer ID: 94073
Keywords: hiPSC (Human induced pluripotent stem cells), Immunocytochemistry, Immunofluorescence, Neural differentiation, Motor neuron differentiation
Abstract
This immunocytochemistry protocol is used for the characterization of IPSC differentiation into motor neurons using several biomarkers: neuroepithelial cells (SOX1), motor neuron progenitors (OLIG2 and NKX2.2), motor neurons (MNX1), and the mature motor neurons (ISL, ChAT, MAP2).

*Primary and secondary antibody information located in materials section





Materials
ReagentBlockAid™ Blocking SolutionThermo FisherCatalog #B10710
Reagent Phosphate-buffered saline (PBS, 1X), sterile-filteredThermo ScientificCatalog #J61196.AP
ReagentTriton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML
ReagentBovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A4612
Reagent32% ParaformaldehydeElectron Microscopy SciencesCatalog #50-980-495

Primary Antibody Stains and Concentrations

Step 1:

  • SOX1 (R+D Systems - AF3369 - 100 ug) - Reconstitute in 500uL of sterile 1xPBS makes 200 ug/ul concentration - Use
15 ug/ml ***Add 75ul to 1ml BSA*** - Needs Anti-Goat Secondary
Step 2:
  • OLIG2 (Sigma - AB9610-100ul volume in tube-0.5 mg/ml,) - Use 1.2 ug/mL (1:400 dilution)
***Add 2.5ul to 1ml BSA*** - Needs Anti-Rabbit Secondary

  • NKX2 (DSHB - 74.5A5 - 1ml total volume in tube - 23 ng/ul) - Use 2ug/ml total
***Add 86ul to 1ml BSA*** - Needs Anti-Mouse Secondary
Step 4:

  • MNX1 (DSHB - 81.5C10 - 1ml total volume in tube - 36 ng/ul) - Use 2ug/ml total
***Add 55ul to 1ml BSA*** - Needs Anti-Mouse Secondary
  • MNX1 (2nd option) (Novus Biological- NBP224691- 0.1 mg/ml) - Use 2ug/ml total
 ***Add 4ul to 1mL BSA***- Needs Anti-Rabbit Secondary
Step 5:

  • MAP2 (Sigma - M3696-100ug in tube - 1.0 mg/mL ) - Use 2.5 ug/mL (1:400 dilution)
***Add 2.5ul to 1ml BSA*** - Needs Anti-Rabbit Secondary

  • MAP2 (2nd option) (Thermo Scientific- PA5-17646 - 100 uL in tube - 73.6 µg/mL ) - Use 0.74 ug/mL (1:100 dilution) ***Add 10ul to 1ml BSA*** - Needs Anti-Rabbit Secondary
  • CHAT (Sigma - AB144P - 500ul - Concentration: > = 0.1 - < 1%) - Use 1 ug/mL (1:100 dilution)
***Add 10ul to 1ml BSA*** - Needs Anti-Goat Secondary
  • ISL1 (DSHB - 40.2D6 - 1ml total volume in tube - 28 ng/ul) - Use 2 ug/ml total
***Add 70ul to 930ul BSA*** - Needs Anti-Mouse Secondary


Secondary Antibody Stains and Concentrations
(Use volumes are based on a total volume of 1ml 3% BSA staining solution. Volumes of Primary or Secondary antibodies should be subtracted from 1ml volume, ie. 150ul SOX1 added to 850ul BSA = 1ml BSA total.  Adjust volumes as needed for staining solutions)
  • Alexa Fluor Plus 488 Donkey Anti-Rabbit IgG (H+L) (ThermoFisher A32790- 1 mg in tube - 2 mg/mL stock) -
- Use 2 ug/ml total ***Add 1ul to 1ml BSA***
  • Alexa Fluor 555 Donkey Anti-Goat IgG (H+L) (ThermoFisher A21432 - 1 mg in tube - 2 mg/ml stock)
- Use 10 ug/ml total ***Add 5ul to 1ml BSA***
  • AlexaFluor 647 Donkey Anti-Mouse IgG (H+L) (ThermoFisher A31571 - 1mg in tube - 2 mg/ml stock)
- Use 2 ug/ml total ***Add 1ul to 1ml BSA***
Before start
To ensure that the observed results are not just random events, use controls, such as undifferentiated IPSCs when doing immunocytochemistry.

Here is an example of how we seed cells onto a 96-well plate. The plate has an equal amount of wells of differentiated (blue) and undifferentiated cells (yellow) for analysis, and we always include a moat (evaporation barrier). The cells are also counted and seeded at equal densities.

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A
B
Diff
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IPSC
IPSC
C
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IPSC
IPSC
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IPSC
IPSC
E

Remove the medium from your cells
Note
Tip the vessel towards you and pipette from the bottom corner of the well





FIX - Dilute 32% Paraformaldehyde solution to 4% PFA in 1X phosphate-buffered saline (PBS)
Add 100uL of 4% PFA to each well in the 96-well plate. (1ml if using a 6-well plate). Incubate for Duration00:15:00 at room temperature.

15m
Remove the fixative solution and wash with 1XPBS at 100ul per well using a multichannel pipette. Repeat 3 times.
Note
  • Aspirate and dispense fixative and wash very slowly, so as to not dislodge your cells.
  • Move the plate from side to side between each wash
  • The fixed sample can be stored, covered in foil, for several days at 4°C if needed.

PERMEABILIZE - Add 100uL of 0.5% Triton X-100 to each well of a 96-well (1ml if using 6-well plate)
Incubate for Duration00:15:00 at room temperature.

15m
Remove the permeabilization solution and wash 3 times with 1XPBS
BLOCK - Add 100ul of 3% BSA (bovine serum albumin) or blockAid-blocking solution to each well of a 96-well plate slowly to Block. (1ml if using 6-well plate)
Incubate for at leastDuration01:00:00 (up to overnight) at room temperature.

1h
Calculate the amount of primary antibody needed (located in materials section) and dilute in 3% BSA + 0.3% Triton X-100 solution
PRIMARY ANTIBODIES - Remove 3% BSA from wells and add 100uL of primary antibody per well

Note
Make sure primary and secondary antibodies are stored properly. Aliquots should be put in the -20 and thawed once, with any remainder kept at 4°C.

Incubate for Duration01:00:00 at room temperature in a dark place or overnight at 4°C.
(Different primary antibody stains may take longer to stain and could require optimization).
1h
Remove primary antibody and wash three times slowly with 1xPBS
Note
  • Remember to aspirate and dispense fixative and wash very slowly, so as to not dislodge your cells and continue moving plate side to side between washes.


SECONDARY ANTIBODIES - Calculate amount of secondary antibody needed (located in materials section) and dilute in 3% BSA+ .3% TritonX-100 solution and 1:4000 Hoechst.
Add 100uL per well in 96-well and incubate for at least Duration01:00:00 at room temperature in a dark place.

Remove secondary antibody and wash three times with 1xPBS
Scan on the confocal microscope
Note
We currently use the ImageXpress Confocal HT.ai High-Content imaging system and the InCarta image analysis software to asses the presence and intensity of specific protein and to quantify the percent of live nuclei.



Expected result




Neuroepithelial cells stained with SOX1/anti-goat (shown as green in photo) and Hoechst (blue)

Neural progenitors stained with Olig2/anti-rabbit (green) and Hoechst (blue)



Motor neurons stained with MNX1/anti-mouse (red) and Hoechst (blue)


Mature motor neurons stained with ISL/anti-mouse (red) and Hoechst (blue)



Mature motor neurons stained with Map2/anti-rabbit (green) and Hoechst (blue)





Protocol references
Du, ZW., Chen, H., Liu, H. et al. Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells. Nat Commun 6, 6626 (2015). https://doi.org/10.1038/ncomms7626