Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes

Summary

We present a new fluorescence technique for selective in situ labeling of active phagocytic cells, which clear off cell corpses in stroke. The approach is important for assessing brain reaction to ischemia because only a small proportion of phagocytes present in ischemic brain participate in clearance of cell death.

Abstract

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination of dead cells in stroke by waste-management phagocytes of any cellular lineage. Although numerous cells of different origins that are capable of phagocytosis are present in ischemic brain, only part of them actively engulf and digest cell corpses. The selective visualization, quantification and analysis of such active phagocytic waste-management are helpful in assessing brain response to ischemia. Efficient cell death clearance is important for brain recovery from ischemic injury, as it opens the way for the subsequent regenerative processes. The failure to clean the corpses would result in a toxic reaction caused by non-degraded DNA and proteins. The described procedure uses fluorescent probes selectively ligated by a viral topoisomerase to characteristic DNA breaks produced in all phagocytes during engulfment and digestion of cells irreversibly damaged by ischemia. The method is a new tool for the investigation of brain reaction to ischemic injury.

Introduction

Ischemic stroke induces profound changes in the affected brain tissue. It initiates massive cell death, which leads to the rapid activation of resident phagocytic cells of microglial origin. It also sets off infiltration of ischemic brain by various types of blood-derived professional phagocytes including neutrophils, macrophages, dendritic, and mast cells^1,2.

It is still debated whether this response to ischemic injury plays a positive or a negative role. Although phagocytosis following stroke can be beneficial because it clears dead cells and suppresses inflammation, it also generates toxic reactive oxygen species affecting neu....

Protocol

The method is suitable for formaldehyde-fixed, paraffin embedded tissue sections. The technique is demonstrated here in its application to experimental stroke in rat.

1. Preparing Components for Tyramide Signal Amplification (TSA) System

Although the TSA-based enhancement is used in the final stage of labeling, preparing for this reaction can take more than 1 hr. Therefore it is convenient to make the TSA reagents before starting the labeling.

1. Make fluorophore tyramide stock solution. Use the fluorophore tyramide reagent supplied in individual vials with the TSA labeling kit. Add 300 µl DMSO....

Results

The principle of the technique for labeling active waste-management phagocytes is presented in Figure 1. The schematic demonstrates that the detection proceeds in three steps. The first step encompasses probe activation (Figure 1A); in the second step the activated probe is ligated to specific DNA breaks in tissue sections (Figure 1C); the third step includes fluorescent signal amplification (Figure 1D)........

Discussion

In this video we demonstrate, in the tissue section format, how to label active phagocytes which clear cell death in focal brain ischemia. The presented technique is the first approach which specifically labels only those waste-management cells which have engulfed and actively digest cellular corpses. This makes it advantageous with respect to other existing methods for labeling of phagocytic cells, which do not have this capability.

The method uses a general and selective DNA-based marker of.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Xylene	JT Baker	9490-22
Ethanol
Proteinase K	Roche	3115879001
PBS Buffer	Invitrogen	AM9624
Vaccinia Topoisomerase I	Vivid Technologies		5 pmol/μl stock
12-Base Overhang Suicide Oligonucleotide	IDT DNA		5’-AAG GGA CCT GCB GCA GGT CCC TTG ATA CGA TTC TA -3’; B – Biotin-dT; 100 pmol/μl stock in ddH2O
Adapter Oligonucleotide	IDT DNA		5’-TAG AAT CGT ATC-3’; 100 pmol/μl stock in ddH2O
1 M Tris-HCl, PH 7.4	Lonza	51237
TSA, Fluorescein System	PerkinElmer	NEL701A001KT
1 M NaCl
DMSO	Sigma
Tween-20	Sigma	P9416-50ML
Vectashield with DAPI	Vector Laboratories	H-1200
Coverslips			22 x 22 mm or 22 x 40 mm glass or plastic coverslips. Plastic coverslips are preferable during the reaction, as they are easier to remove from the section.
Rat Brain Sections			Rat brain taken 48 hr after reproduction of ischemic stroke as described in Didenko et al11. 5-6 μm-thick sections cut from paraformaldehyde-fixed, paraffin-embedded tissue blocks. Use charged and precleaned slides.
Inverted Microscope	Olympus
Digital Video camera and software	Micromax
Software	Metamorph