TITLE: Immunofluorescence of macrophages and microglia
AUTHORS: Narayana Yadavalli, Shawn M. Ferguson

[DESCRIPTION]
This protocol describes the fixation and staining of cultured cells in paraformaldehyde solution.

[STEPS]
SECTION: Immunofluorescence of macrophages and microglia
1. Plate 50,000 iPSC derived macrophages or microglia or Neurons or BMDM’s cells on 22 x 22 mm glass coverslips in 24-well dishes in respective culture media.
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2. Incubate   at 37 °C in 5% CO2.

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3. Pre-warm a solution of 4% paraformaldehyde to Room temperature.

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4. Aspirate media from cells and immediately proceed to step 5.
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5. Add pre-warmed 4% paraformaldehyde to cells.
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6. Incubate for 15 min at Room temperature.

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7. Remove 4% paraformaldehyde solution and dispose of it in an appropriate chemical waste container.
SECTION: Immunofluorescence of macrophages and microglia
8. Rinse each well with 3X 1 mL PBS. Remove rinse and dispose of it in an appropriate chemical waste container.

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9. Add 0.1% (v/v) saponin + 5% (w/v) BSA in PBS and incubate for 60 min at Room temperature to permeabilize cells.

SECTION: Immunofluorescence of macrophages and microglia
10. Aspirate 0.1% (v/v) saponin + 5% (w/v) BSA in PBS solution.
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11. Prepare respective antibody dilutions in 0.1% (v/v) saponin + 5% (w/v) BSA in PBS solution. mouse LAMP1 (DSHB #ID4B) 1:200 dilution, TFE3 (Sigma #HPA023881), 1:200 dilution.
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12. Incubate at 4 °C 60 min.

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13. Aspirate antibody solution and wash cells with PBS for 5 min.

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14. Repeat wash.
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15. Add filtered PBS containing 5% BSA and secondary antibodies (1:600, Alexa fluorophores 488 and 555, Invitrogen).
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16. Incubate at Room temperature for 60 min.

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17. Aspirate antibody solution and wash cells.
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18. Rinse coverslips in milliQ water.
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19. Mount coverslips onto slides using ProLong Gold Antifade Mountant with DAPI (ThermoFisher P36935).
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20. Allow slides to cure 5 min in darkness.

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21. Image.
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14.1. Wash with PBS for 5 min. (1/3)

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14.2. Wash with PBS for 5 min.(2/3)
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14.3. Wash with PBS for 5 min.(3/3)
SECTION: Immunofluorescence of macrophages and microglia
17.1. Wash cells with PBS for 5 min. (1/3)

SECTION: Immunofluorescence of macrophages and microglia
17.2. Wash cells with PBS for 5 min. (2/3)
SECTION: Immunofluorescence of macrophages and microglia
17.3. Wash cells with PBS for 5 min. (3/3)