TITLE: Immunocytochemistry of motor neurons derived from iPSCs with the hNIL construct protocol
AUTHORS: Maria  Sckaff, Carissa Feliciano, Zachary Nevin, Bruce  Conklin, Claire D Clelland

[DESCRIPTION]
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.

[STEPS]
SECTION: Immunocytochemistry of the hNIL motor neurons: Day 1: Fixing, Permeabilizing, Blocking and Coating Cells with Primary Antibody
1. 
 
Without removing media from the wells, add 4% PFA on all target wells and let it sit for 30 min at Room 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).

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

SECTION: Immunocytochemistry of the hNIL motor neurons: Day 1: Fixing, Permeabilizing, Blocking and Coating Cells with Primary Antibody
3. Wash cells with 1X DPBS + 0.1% Triton-X (“DPBS-T” at 100 µL per well) to permeabilize the cells. 
SECTION: Immunocytochemistry of the hNIL motor neurons: Day 1: Fixing, Permeabilizing, Blocking and Coating Cells with Primary Antibody
4. Block the target wells with 1X DPBS-T + 5% BSA at 4 Room temperature for 60 min (50 µL/well). 
 


SECTION: Immunocytochemistry of the hNIL motor neurons: Day 1: Fixing, Permeabilizing, Blocking and Coating Cells with Primary Antibody
5. Discard blocking agent onto an absorbent wipe, then add primary antibodies diluted in 1X DPBS-T + 5% BSA (50 µL/well) at the appropriate concentrations.
 

SECTION: Immunocytochemistry of the hNIL motor neurons: Day 1: Fixing, Permeabilizing, Blocking and Coating Cells with Primary Antibody
6. Incubate at 4 °C 60 min on a plate shaker, with the sides covered by parafilm to avoid evaporation.

SECTION: Immunocytochemistry of the hNIL motor neurons: Day 1: Fixing, Permeabilizing, Blocking and Coating Cells with Primary Antibody
3.1. Briefly wash cells with 1X DPBS + 0.1% Triton-X (“DPBS-T” at 100 µL per well). Discard the spent wash onto absorbent wipes. (1/3)
SECTION: Immunocytochemistry of the hNIL motor neurons: Day 1: Fixing, Permeabilizing, Blocking and Coating Cells with Primary Antibody
3.2. Wash cells with 1X DPBS + 0.1% Triton-X (“DPBS-T” at 100 µL per well) for 10 min at slow agitation on the Belly Dancer. Discard the spent wash onto absorbent wipes. (2/3)

SECTION: Immunocytochemistry of the hNIL motor neurons: Day 1: Fixing, Permeabilizing, Blocking and Coating Cells with Primary Antibody
3.3. Wash cells with 1X DPBS + 0.1% Triton-X (“DPBS-T” at 100 µL per well) for 10 min at slow agitation on the Belly Dancer.  Discard the spent wash onto absorbent wipes. (3/3)
SECTION: Immunocytochemistry of the hNIL motor neurons: Day 2: Coating Cells with Secondary Antibody
7. Discard the primary antibody solution from each well using a multichannel pipette.
 

SECTION: Immunocytochemistry of the hNIL motor neurons: Day 2: Coating Cells with Secondary Antibody
8. Wash with 1X DPBS-T to remove any unbound primary antibody (100 µL/well). First briefly, then twice for 10 minutes each time at slow agitation on the Belly Dancer.

SECTION: Immunocytochemistry of the hNIL motor neurons: Day 2: Coating Cells with Secondary Antibody
9. Add desired secondary antibodies diluted in 1X DPBS-T + 5% BSA. Incubate at4 Room temperature for 60 min in the belly dancer.

SECTION: Immunocytochemistry of the hNIL motor neurons: Day 2: Coating Cells with Secondary Antibody
10. Wash once briefly with 1X DPBS-T (100 µL/well).

SECTION: Immunocytochemistry of the hNIL motor neurons: Day 2: Coating Cells with Secondary Antibody
11. Wash with 1X DPBS-T with DAPI (1:1000-1:10,000) for 10 min (100 μL/well).
SECTION: Immunocytochemistry of the hNIL motor neurons: Day 2: Coating Cells with Secondary Antibody
12. Wash with 1X DPBS (100 μL/well).
SECTION: Immunocytochemistry of the hNIL motor neurons: Day 2: Coating Cells with Secondary Antibody
13. Change the media to 1X DPBS or 1X DPBS + Sodium Azide 0.02%, if storing long term, (100 µL/well) and store at 4 °C.
SECTION: Immunocytochemistry of the hNIL motor neurons: Day 2: Coating Cells with Secondary Antibody
8.1. Wash with 1X DPBS-T briefly. (1/3)
SECTION: Immunocytochemistry of the hNIL motor neurons: Day 2: Coating Cells with Secondary Antibody
8.2. Wash with 1X DPBS-T for 10 min at slow agitation on the Belly Dancer. (2/3)

SECTION: Immunocytochemistry of the hNIL motor neurons: Day 2: Coating Cells with Secondary Antibody
8.3. Wash with 1X DPBS-T for 10 min at slow agitation on the Belly Dancer. (3/3)
SECTION: Immunocytochemistry of the hNIL motor neurons: Day 2: Coating Cells with Secondary Antibody
12.1. Wash with 1X DPBS for 10 min. (1/2)

SECTION: Immunocytochemistry of the hNIL motor neurons: Day 2: Coating Cells with Secondary Antibody
12.2. Wash with 1X DPBS for 10 min. (2/2)