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

Published: April 30, 2023

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 Immunostaining with an anti-Croquemort antibody 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. Mount 20 µL of 5% (v/v) whole swine serum in PBS containing 0.2% (v/v) Triton X-100 on cells for blocking. …

Divulgations

The authors have nothing to disclose.

Materials

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

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The Phagocytosis Assay: An Immunostaining Technique to Visualize Apoptotic Cell Engulfment in the Embryonic Cells of Drosophila melanogaster. J. Vis. Exp. (Pending Publication), e21235, doi: (2023).

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