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

Abstract

Source: Nonaka, S. et al. Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos. J. Vis. Exp. (2017).

This video demonstrates an immunostaining technique to visualize apoptotic cell engulfment in the homogenized embryonic cells of Drosophila melanogaster. This method provides insights into cellular physiology by determining the extent of apoptotic cell engulfment.

Protocol

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....

Materials

List of materials used in this article

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