The Phagocytosis Assay: An Immunostaining Technique to Visualize Apoptotic Cell Engulfment in the Embryonic Cells of Drosophila melanogaster

Protocole

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Staining of Hemocytes

1. Immunostaining with an anti-Croquemort antibody
   1. Serially soak glass slides in methanol for 10 min, in PBS containing 0.2% (v/v) Triton X-100 for 10 min, and then in PBS for 10 min.
   2. Mount 20 µL of 5% (v/v) whole swine serum in PBS containing 0.2% (v/v) Triton X-100 on cells for blocking. Incubate slides at room temperature for 20 min.
   3. Remove the solution from glass slides, and mount 20 µL of anti-Croquemort antiserum (0.1% (v/v)) in PBS containing 0.2% (v/v) Triton X-100 on cells. Incubate slides at 4 °C overnight.
   4. Soak glass slides in PBS containing 0.2% (v/v) Triton X-100 for 10 min 5 times.
   5. Soak glass slides in PBS for 10 min.
   6. Mount 20 µL of alkaline phosphatase-labeled anti-rat IgG (0.5% (v/v)) in PBS containing 0.2% (v/v) Triton X-100 and 5% (v/v) whole swine serum on cells at room temperature for 1 h.
   7. Soak glass slides in PBS containing 0.2% (v/v) Triton X-100 for 10 min 5 times.
   8. Soak glass slides in buffer containing 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 50 mM MgCl2 for 10 min.
   9. Mount phosphatase substrate solution containing 0.23 mg/mL 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), 0.35 mg/mL nitro blue tetrazolium (NBT), 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 50 mM MgCl₂ on cells.
   10. Observe cells under a microscope for proper staining. When strong purple signals appear in the granules of hemocytes, remove the substrate solution, and soak glass slides in buffer consisting of 10 mM Tris-HCl, pH 8.5, and 1 mM EDTA. Staining for approximately 5 - 10 min is recommended.

2. Stain Apoptotic Cells by TUNEL

1. Soak glass slides in PBS for 5 min.
2. Repeat with new PBS.
3. Mount Equilibration buffer (See Materials Table) on cells for 10 min.
4. Remove the solution from glass slides, and mount 20 µL of TdT solution containing 5 µL of terminal deoxynucleotidyl transferase and 15 µL of Reaction Buffer on cells at 37 °C for 1 h.
5. Remove the solution from glass slides, and soak slides in buffer consisting of 0.5 mL STOP/Wash buffer and 17 mL of water.
6. Soak slides in PBS for 5 min 3 times.
7. Mount 20 µL of Anti-Digoxigenin-Peroxidase on cells at room temperature for 30 min.
8. Soak glass slides in PBS for 5 min 4 times.
9. Soak glass slides in peroxidase substrate solution consisting of 30 mL of 50 mM Tris-HCl, pH7.5 containing a full micro spatula of 3,3'-diaminobenzidine tetrahydrichloride (DAB), and 0.002% (v/v) H₂O₂ for 30 s.
10. Repeat the above step until apoptotic cells are stained brown. Soak glass slides in water to stop the peroxidase reaction.
11. Enclose cells with water for observation.

3. Measure the Level of Phagocytosis of Apoptotic Cells

1. Observe samples under a light microscope in order to assess the level of phagocytosis. Count Croquemort-positive cells or GFP-positive cells as hemocytes, TUNEL-positive cells as apoptotic cells, and double-positive cells as phagocytosing hemocytes.
2. The phagocytic index is defined as the number of double-positive cells to the total number of Croquemort-positive cells or GFP-positive cells. It is recommended that more than 300 hemocytes are observed.

matériels

Name	Company	Catalog Number	Comments
whole swine serum	MP Biomedicals	55993	For blocking
Kpl Anti-Rat IgG (H+L) Ab MSA, AP	KPL	475-1612	secondary antibody for stainig hemocytes with an anti-Croquemort antibody
5-bromo-4-chloro-3-indolyl-phosphate	Roche	11383221001	BCIP, For staining of hemocytes
nitro blue tetrazolium	Roche	11383213001	NBT, For staining of hemocytes
Anti-Croquemort antibody			described previously in Manaka et al, J. Biol. Chem., 279, 48466-48476
Apop Tag Peroxidase In Situ Apoptosis Detection Kit	Millipore	S7100	For staining of apoptoitc cells. This kit includes Equilibration buffer, Reaction buffer, STOP/Wash buffer, TdT enzyme, and Anti-Digoxigenin-Peroxidase.
3,3'-diaminobenzidine tetrahydrichloride	nacalai tesque	11009-41	DAB, For staining of apoptoitc cells