Immunocytochemistry of motor neurons derived from iPSCs with the hNIL construct protocol
Maria Sckaff, Bruce Conklin, Claire D Clelland, Carissa Feliciano, Zachary Nevin
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
Steps
Immunocytochemistry of the hNIL motor neurons: Day 1: Fixing, Permeabilizing, Blocking and Coating Cells with Primary Antibody
0h 30m 0s 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). 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.
Wash cells with 1X DPBS + 0.1% Triton-X (“DPBS-T” at 100µL per well) to permeabilize the cells.
Briefly wash cells with 1X DPBS + 0.1% Triton-X (“DPBS-T” at 100µL per well). Discard the spent wash onto absorbent wipes. (1/3)
Wash cells with 1X DPBS + 0.1% Triton-X (“DPBS-T” at 100µL per well) for 0h 10m 0s at slow agitation on the Belly Dancer. Discard the spent wash onto absorbent wipes. (2/3)
Wash cells with 1X DPBS + 0.1% Triton-X (“DPBS-T” at 100µL per well) for 0h 10m 0s at slow agitation on the Belly Dancer. Discard the spent wash onto absorbent wipes. (3/3)
Block the target wells with 1X DPBS-T + 5% BSA at 4Room temperature for 1h 0m 0s (50µL/well).
Discard blocking agent onto an absorbent wipe, then add primary antibodies diluted in 1X DPBS-T + 5% BSA (50µL/well) at the appropriate concentrations.
Incubate at 4°C 1h 0m 0s on a plate shaker, with the sides covered by parafilm to avoid evaporation.
Immunocytochemistry of the hNIL motor neurons: Day 2: Coating Cells with Secondary Antibody
Discard the primary antibody solution from each well using a multichannel pipette.
Wash with 1X DPBS-T to remove any unbound primary antibody (100µL/well). First briefly, then twice for 10 minutes each time at slow agitation on the Belly Dancer.
Wash with 1X DPBS-T briefly. (1/3)
Wash with 1X DPBS-T for 0h 10m 0s at slow agitation on the Belly Dancer. (2/3)
Wash with 1X DPBS-T for 0h 10m 0s at slow agitation on the Belly Dancer. (3/3)
Add desired secondary antibodies diluted in 1X DPBS-T + 5% BSA. Incubate at4Room temperature for 1h 0m 0s in the belly dancer.
Wash once briefly with 1X DPBS-T (100µL/well).
Wash with 1X DPBS-T with DAPI (1:1000-1:10,000) for 0h 10m 0s (100 μL/well).
Wash with 1X DPBS (100 μL/well).
Wash with 1X DPBS for 0h 10m 0s. (1/2)
Wash with 1X DPBS for 0h 10m 0s. (2/2)
Change the media to 1X DPBS or 1X DPBS + Sodium Azide 0.02%, if storing long term, (100µL/well) and store at 4°C.
