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. …
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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 |
