May 16, 2022

Public workspaceImmunocytochemistry of motor neurons derived from iPSCs with the hNIL construct protocol

DOI
dx.doi.org/10.17504/protocols.io.6qpvr68bovmk/v1
  • 1University of California, San Francisco;
  • 2Gladstone Institutes, San Francisco, CA, United States;
  • 3UCSF Weill Institute for Neurosciences
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DOI: dx.doi.org/10.17504/protocols.io.6qpvr68bovmk/v1
Protocol Citation: Maria Sckaff, Carissa Feliciano, Zachary Nevin, Bruce Conklin, Claire D Clelland 2022. Immunocytochemistry of motor neurons derived from iPSCs with the hNIL construct protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr68bovmk/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: March 24, 2022
Last Modified: May 16, 2022
Protocol Integer ID: 59858
Keywords: hNIL motor neurons, immunocytochemistry staining, pluripotent stem cells (iPSCs), ICC, immunofluorescence staining, motor neuron staining, immunofluorescence
Abstract
This protocol describes the immunocytochemistry for staining motor neurons derived from induced pluripotent stem cells (iPSCs) using the hNIL transgenic factors in a CLYBL safe harbor site. For the protocol on this differentiation, refer to the Clelland Lab’s Differentiation of iPSCs with the hNIL construct into motor neurons protocol.
Attachments
Icon for ICC of motor neurons derived from iPSCs with the hNIL construct_ClellandLab.pdf
ICC of motor neurons...
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Materials

Equipment used in the immunocytochemistry staining of motor neurons derived from hNIL iPSCs.
ABC
EquipmentManufacturerCatalog Number
Falcon® 96-well Black/Clear Flat Bottom TC-treated Imaging Microplate with LidCorning353219
Stovall Life Science, The Belly DancerFisher Scientific15-453-211
VWR Mini ShakerAvantor/VWR12620-938

Reagents used in the immunocytochemistry staining of motor neurons derived from hNIL iPSCs.
ABC
EquipmentManufacturerCatalog Number
4% PFAAlfa AesarJ61899
1X DPBS, no Ca, no MgThermoFisher14-190-235
Bovine Serum Albumin (BSA)Sigma AldrichA4503-50G
Triton X-100anyany
DAPI (4'-6-Diamidino-2-Phenylindole, Dihydrochloride)ThermoFisherD1306


Equipment
6-well Black/Clear Flat Bottom TC-treated Imaging Microplate with Lid
NAME
Microplate
TYPE
Falcon®
BRAND
353219
SKU
https://ecatalog.corning.com/life-sciences/b2c/US/en/Microplates/Assay-Microplates/96-Well-Microplates/Falcon%C2%AE-96-well-Polystyrene-Microplates/p/353219?pagePath=p/353219
LINK

Equipment
The Belly Dancer™ Orbital Platform Shaker
NAME
Shaker
TYPE
IBI Scientific™
BRAND
15-453-211
SKU
https://www.fishersci.com/shop/products/ibi-scientific-the-belly-dancer-shakers-lab-start-up-kit-extension-platform/15453211
LINK

Equipment
Mini Shakers
NAME
Shakers
TYPE
VWR®
BRAND
12620-938
SKU
https://us.vwr.com/store/product/4902576/vwr-mini-shakers
LINK
Other suggested materials:
  • Parafilm
  • Sodium Azide
Reagentbeta Tubulin Monoclonal Antibody (AA10)Thermo Fisher ScientificCatalog #480011

Immunocytochemistry of the hNIL motor neurons: Day 1: Fixing, Permeabilizing, Blocking and Coating Cells with Primary Antibody
Immunocytochemistry of the hNIL motor neurons: Day 1: Fixing, Permeabilizing, Blocking and Coating Cells with Primary Antibody
2h 50m
2h 50m

Note
This protocol can be followed at any time after Day 7 in the hNIL differentiation of iPSCs into motor neurons protocol. This protocol has been optimized to neurons plated on 96-well plates.
Without removing media from the wells, add 4% PFA on all target wells and let it sit for Duration00:30:00 at TemperatureRoom temperature (the volume of 4% PFA should equal the volume of media already in the well, resulting in a final well concentration of 2% PFA).

30m
Discard the media and PFA by carefully and gently tapping the plate upside down onto absorbent wipes (KIMTECH Kimwipes are appropriate). Discard the Kimwipes in designated PFA waste.
Note
If fixing and staining need to be performed on separate days, after step 2, wash the plate thrice with 1X DPBS, Duration00:10:00 each time at slow agitation on the Belly Dancer. Then store the plate at Temperature4 °C , with the sides covered by parafilm to avoid evaporation. Only permeabilize the cells (step 3) on the day that the cells will be incubated with primary antibody.


Wash cells with 1X DPBS + 0.1% Triton-X (“DPBS-T” at Amount100 µL per well) to permeabilize the cells.
Wash
Briefly wash cells with 1X DPBS + 0.1% Triton-X (“DPBS-T” at Amount100 µL per well). Discard the spent wash onto absorbent wipes. (1/3)
Wash cells with 1X DPBS + 0.1% Triton-X (“DPBS-T” at Amount100 µL per well) for Duration00:10:00 at slow agitation on the Belly Dancer. Discard the spent wash onto absorbent wipes. (2/3)

10m
Wash cells with 1X DPBS + 0.1% Triton-X (“DPBS-T” at Amount100 µL per well) for Duration00:10:00 at slow agitation on the Belly Dancer. Discard the spent wash onto absorbent wipes. (3/3)
10m
Block the target wells with 1X DPBS-T + 5% BSA at TemperatureRoom temperature for Duration01:00:00 (Amount50 µL /well).
Note
Do not exceed this time to avoid over blocking your neurons.



1h
Critical
Discard blocking agent onto an absorbent wipe, then add primary antibodies diluted in 1X DPBS-T + 5% BSA (Amount50 µL /well) at the appropriate concentrations.
Note
It is advisable to include the beta tubulin monoclonal primary antibody (1:250 from ThermoFisher Scientific Catalog # 480011) to show neuronal morphology.


Incubate at Temperature4 °C DurationOvernight on a plate shaker, with the sides covered by parafilm to avoid evaporation.

1h
Incubation
Overnight
Immunocytochemistry of the hNIL motor neurons: Day 2: Coating Cells with Secondary Antibody
Immunocytochemistry of the hNIL motor neurons: Day 2: Coating Cells with Secondary Antibody
1h 50m
1h 50m
Discard the primary antibody solution from each well using a multichannel pipette.
Note
Ensure to never touch the bottom of the well and always pipette slowly to not lift neurons from the well bottom.

Critical
Wash with 1X DPBS-T to remove any unbound primary antibody (Amount100 µL /well). First briefly, then twice for 10 minutes each time at slow agitation on the Belly Dancer.

Wash
Wash with 1X DPBS-T briefly. (1/3)
Wash with 1X DPBS-T for Duration00:10:00 at slow agitation on the Belly Dancer. (2/3)

10m
Wash with 1X DPBS-T for Duration00:10:00 at slow agitation on the Belly Dancer. (3/3)
10m
Add desired secondary antibodies diluted in 1X DPBS-T + 5% BSA. Incubate atTemperatureRoom temperature for Duration01:00:00 in the belly dancer.

1h
Wash once briefly with 1X DPBS-T (Amount100 µL /well).

Wash
Wash with 1X DPBS-T with DAPI (1:1000-1:10,000) for Duration00:10:00 (100 μL/well).
10m
Wash
Wash with 1X DPBS (100 μL/well).
Wash
Wash with 1X DPBS for Duration00:10:00 . (1/2)

10m
Wash with 1X DPBS for Duration00:10:00 . (2/2)
10m
Change the media to 1X DPBS or 1X DPBS + Sodium Azide 0.02%, if storing long term, (Amount100 µL /well) and store at Temperature4 °C .