Single and Dual Birthdating Procedures for Assessing the Response of Adult Neural Stem Cells to the Infusion of a Soluble Factor Using Halogenated Thy

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

The factors that regulate the switch from adult neural stem cell (aNSC) quiescence to active proliferation are poorly understood. Here we describe a method to study the in vivo effect of a soluble factor on cell cycle entry and proliferation of aNSCs located in the brain neurogenic niches. First, we provide information for implanting osmotic minipumps that will deliver the compound of interest directly into the mouse brain. When combined with the administration of the thymidine analog bromodeoxyuridine (BrdU), this technique is the most basic procedure to study the effects of a soluble factor on aNSC proliferation. We also describe a dual replication labeling protocol using two different halogenated thymidine analogs, chloro? and iododeoxyuridine (CldU and IdU), that allows tracking of proliferating cells and assessing cell cycle re?entry of aNSCs at different time points. Curr. Protoc. Stem Cell Biol. 21:2D.10.1?2D.10.20. © 2012 by John Wiley & Sons, Inc.

Keywords: neural stem cell; label?retaining cell; IdU; CldU; BrdU; intracerebroventricular infusion; osmotic minipump

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Introduction
Basic Protocol 1: Single Labeling of Actively Proliferating Cells to Analyze the Short‐Term Effect of a Soluble Factor on aNSCs
Basic Protocol 2: Dual Labeling of Two Cell Division Rounds to Analyze the Long‐Term Effect of a Soluble Factor on aNSCs
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Single Labeling of Actively Proliferating Cells to Analyze the Short‐Term Effect of a Soluble Factor on aNSCs

Materials

Mice: 2‐month‐old Crl:CD1(ICR) male mice (Charles River Laboratories, strain code 22)
Factor or drug of interest
Sterile saline: 0.9% (w/v) NaCl
70% ethanol
Ketamine/xylazine anesthetic (see recipe )
Meloxicam analgesic (see recipe )
Surgical glue: Hystoacryl (B. Braun Biosurgical; http://www.bbraun.com/)
10 mg/ml BrdU (see recipe for thymidine analogs)
IsobaVet (100% isoflurane; Schering‐Plough; store at room temperature)
4% paraformaldehyde (PFA; see recipe )
0.1 M PB (see recipe ) with and without 0.05% sodium azide, 4°C
Cyanoacrylate‐based fast‐acting glue (e.g., Super Glue)
2% to 3% agar (see recipe ), warm
Cryoprotectant solution (see recipe )
2 N HCl
0.1 N sodium borate, pH 8.5 (see recipe )
Blocking solution (see recipe )
Primary antibody: mouse anti‐BrdU antibody (Dako, cat. no. M0744)
Secondary antibody: Alexa 488‐conjugated donkey anti‐mouse antibody (Invitrogen, cat. no. A21202)
10 µg/ml DAPI working solution (see recipe )
Mowiol (see recipe )

Osmotic minipumps (ALZET Model 1007D for a 7‐day delivery period, 0.5 µl/hr)
Syringe and blunt 27‐G needle for filling minipumps (ALZET)
Brain infusion assembly (ALZET Brain Infusion Kit 3) including:
- Brain infusion cannulae
- Vinyl catheter tubes
- Depth‐adjustment spacers
50‐ml conical polypropylene centrifuge tubes (e.g., BD Falcon)
Surgery tools: large scissors, small pointed scissors, hemostatic forceps, large forceps, sterile scalpels (Fine Science Tools)
Micromotor drill (Leica, cat. no. 39416101)
Steel burr (working diameter between 0.5 and 0.8 mm; Dremel, cat. no. 26150105JA; http://www.dremel.com/)
Stereotaxic apparatus with mouse adaptor (Leica, cat. no. 39477001 and 39462950)
Animal clippers
Thermal blanket (Cibertec, cat. no. RTC1; http://www.cibertec.es/)
Airtight chamber (20 cm high, 5 cm diameter) for IsobaVet administration
BD Vacutainer Push Button Blood Collection Set (ref. no. 367342)
Peristaltic pump (Cole‐Parmer, cat. no. HV‐07524‐40)
100‐mm Petri dishes
Vibratome (Leica VT‐1200‐S)
24‐well tissue culture plates
Soft paint brush
Glass slides and cover slips
Confocal microscope (Leica Spectral SP5) with appropriate lasers for the fluorophores used, and camera

Basic Protocol 2: Dual Labeling of Two Cell Division Rounds to Analyze the Long‐Term Effect of a Soluble Factor on aNSCs

Materials

CldU: 5‐Chloro‐2′‐deoxyuridine (see recipe for thymidine analogs)
IdU: 5‐Iodo‐2′‐deoxyuridine (see recipe for thymidine analogs)
Primary antibodies:
- Monoclonal rat anti‐BrdU antibody (Abcam, cat. no. ab6326) for CldU detection
- Monoclonal mouse anti‐BrdU antibody (BD, cat. no. 7580) for IdU detection
Secondary antibodies:
- Alexa 488‐conjugated donkey anti‐rat (Invitrogen, cat. no. A21208)
- Alexa 594‐conjugated donkey anti‐mouse (Invitrogen, cat. no. A21203)
Alcohol burner

Additional reagents and equipment for single labeling ( protocol 1 )

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Figures

Figure 2.D1.1 Single‐labeling procedure to analyze the short‐term effect of a soluble factor on aNSCs proliferation. (A ) Schematic outline of the experimental design employed for pump implantation, factor delivery, and BrdU injection. (B ) Diagram showing the possible output of BrdU labeling depending on the mode of aNSC division. Active aNSCs can divide symmetrically (left) to generate two aNSCs, expanding the stem cell pool, or asymmetrically (right) to give rise to a stem cell and a linage‐restricted progenitor cell. (C, D ) Confocal images of a triple immunofluorescence showing SOX2⁺ GFAP⁺ aNSCs stained for BrdU in SVZ (C) and SGZ (D). Arrows point at active aNSCs that were proliferating during BrdU administration. Scale bars, 10 µm. GCL, granular cell layer; LV, lateral ventricle; SGZ, subgranular zone; St, striatum.

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Figure 2.D1.2 Preparation of the brain infusion assembly and surgical procedures for the placement of the cannula and ALZET osmotic pump. Preparation of the assembly : (A ) Load the minipump with the solution to be delivered or with sterile saline solution using a syringe. (B ) Assemble the flow moderator, catheter tube, and cannula. Fill the brain infusion assembly with sterile saline solution. (C ) Connect the minipump to the brain infusion assembly, submerge the filled device in sterile saline solution, and incubate at 37°C overnight. Surgical procedures : (D ) Make a rostro‐caudal incision using the scalpel. Prepare a subcutaneous pocket in the back of the mouse. This pocket is created by gently pushing a rounded‐tip scissors from the scalp incision toward the midscapular area. Finally, place the pump in the back of the animal under the skin. (E ) Once the skull is exposed and the periosteal connective tissue has been removed, locate bregma and lambda sutures to determine the site for cannula placement [blue dot, AP 0.0 (0 mm to bregma), L ‐ 0.7 (0.7 mm right from mid‐sagittal line)]. Drill a hole and descend the cannula until the base pedestal reaches the skull. Seal with surgical glue. The stereotaxic coordinates for the lateral ventricle (red dot, corresponding to the final location reached by the tip of the cannula) are AP 0.0, ML ‐ 0.7 and DV 3.0 (3 mm below bregma, determined by the cannula's depth). LV, lateral ventricle; AP, anteroposterior; ML, mediolateral; and DV, dorsoventral.

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Figure 2.D1.3 Dual labeling procedure to analyze the long‐term effect of a soluble factor on aNSC. (A ) Schematic outline of the experimental design employed for infusion and for the administration of CldU and IdU. (B ) Diagram showing the possible labeling of different cell types after the sequential administration of the thymidine analogs. CldU⁺ cells correspond to LRCs, while CldU⁺ cells co‐labeled with IdU⁺ are considered as active LRCs, as they were cycling again during IdU administration. Single IdU⁺ cells are either aNSCs or progenitors that were not cycling during CldU injection. (C, D ) Confocal images of a double CldU/IdU immunofluorescence showing CldU⁺ staining (green), IdU⁺ staining (red), and merge. CldU⁺ cells (arrows) and double‐labeled cells (arrowheads) are shown in the merge panels for the SVZ (C) and SGZ (D). Nuclear stain DAPI is shown blue. Scale bar in (C), 10 µm. Scale bar in (D), 15 µm. SGZ, subgranular zone; Spt, septum; St, striatum.

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Figure 2.D1.4 Verification of the anti‐BrdU antisera specificity in mice injected with CldU only and IdU only. The figure shows confocal images that correspond to the titration in SVZ Vibratome sections of the anti‐BrdU Abcam antibody (for CldU detection) and the BD antibody (for IdU detection). Cross‐reactivity was observed at the starting dilution (1:2000, upper panels). The specific signal for CldU was achieved with the Abcam anti‐BrdU antibody diluted 1:20000. The specific signal for IdU was found with the BD anti‐BrdU antibody diluted 1:5000. Scale bar, 50 µm. LV, lateral ventricle; Spt, septum; St, striatum.

View Image

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Literature Cited

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Key References
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	Original citation of the method described in this unit.