coverslip was secured onto each end of a glassslide with nail varnish. After placing the sliceonto the slide in Citifluor mounting medium(AF1, Agar Scientific), a 40 mm x 22 mm cover-slip was placed to bridge the gap between thetwo 22 mm � 22 mm coverslips, with the slicelying in the 170-µm deep space underneath.The coverslip should be resting gently on thespecimen with little or no gap between thespecimen and the coverslip. The mounts weresealed around the edges of the coverslips withnail varnish and stored in the dark at 4°C for atleast 12 hours to allow the mounting mediumto penetrate evenly throughout the tissue.

Confocal Fluorescence MicroscopyThe fluorescent sections were examined on anOlympus Fluoview FV1000 confocal laser scan-ning microscope using an Olympus UPlanSApo20x NA 0.75 objective lens and 405 nm, 473 nmand 559 nm solid state lasers for excitation.Confocal images were collected and saved inTIFF format using FV10-ASW software (ver01.07.01.00. Olympus). The thickness of wholemount preparations was estimated by scan-ning through the Z-axis of the specimen.When taking optical scans along the Z-axis,gain and offset were optimized for each scan.

RESULTSImmunohistochemistry on the thick wholemount slices initially presented some technicalchallenges including ‘squashing’ of the tissueafter mounting and penetration of antibody.When examining slides on the microscope, itwas observed that the mounted and cover-slipped slices appeared much thinner thantheir predicted thickness of 100 µm [5]. This‘squashing’ of the slice was improved by thebridge mounting technique. The average esti-mated thickness was 30.5 µm with the con-ventional mounting method (n = 7), but 99.83µm with the bridge mounting method (n = 6).

Another anomaly identified in the wholemount preparation was an unlabelled tissue‘blank space’ visible deeper within the slice inthe Z-series scans. When the top was definedas the face of the slice originally in contactwith the atmosphere during culture, and thebottom the membrane side, this ‘blank space’was found to be in the middle of the slice. Thislayer showed little immunoreactivity forMAP2, GFAP, connexin-43 and isolectin-�4(shown for isolectin-�4 in Figure 1 a, b and c).Direct evidence supporting our contentionthat the ‘blank space’ was caused by inade-quate antibody penetration came from serial

B IOGRAPHYDr Jinny Yoon obtainedher BSc (honours) in bio-medical science in 2003,and completed a PhD in2009 at the University ofAuckland. She is cur-rently employed as aresearch technician in the Department ofOphthalmology, where she is involved withthe development of novel therapeuticstrategies to combat degenerative cornealdisorders using adult stem cells.

ABSTRACTThis study aimed to improve existingimmunohistochemical methods in order toobtain continuous multi-fluorescencelabelling throughout the full thickness ofcultured hippocampal slices whilst main-taining structural integrity. Slice cultures,processed as whole mounts for tripleimmunofluorescence labelling, were exam-ined using a confocal laser scanning micro-scope. The thickness of the mounted slicesand penetration of antibodies were investi-gated using conventional and bridgemounting techniques and exposing theslices to various detergent concentrations/durations for permeabilization. It wasshown that a bridge mounting methodafter overnight incubation in 1% Triton X-100 provided high quality immunohisto-chemical data closely reflecting the undis-turbed morphology of slice cultures.

KEYWORDSconfocal fluorescence light microscopy,immunohistochemistry, neuroscience, hip-pocampus, organotypic slice culture

ACKNOWLEDGEMENTSThis research was funded by a Royal Societyof New Zealand Marsden Grant.

AUTHOR DETA I L SDr. Jinny Yoon,Department of Ophthalmology, University of Auckland,Private Bag 92019. Auckland, New Zealand. Tel: +649 373 7599 x87485; Email: j.yoon@auckland.ac.nz

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I N TRODUCT IONOrganotypic hippocampal slice cultures are awidely accepted model system for the rodentcentral nervous system (CNS) [1,2]. Immuno-histochemistry is routinely used to define themorphological characteristics of the slice cul-tures and to quantify the changes in proteinexpression levels. Whole mount immunohisto-chemical labelling preserves the organizationin vivo, allowing imaging of 3D relationshipsbetween cell types and organization of con-nectivity [3]. Detailed immunohistochemistryprotocols are available in the literature [3,4]but none of these really take into account thethickness of the slice when mounting.

This article illustrates some of the drawbacksof current methods for immunohistochemicallabelling of cultured slices and the optimiza-tion steps that can overcome these technicalissues to improve the quality of data obtained.

MATER IALS AND METHODS

Specimen Preparation and StainingThe protocol for preparation of hippocampalslice cultures is described elsewhere [5]. Aftertwo weeks of culture the slices were fixed forone hour in 4% formaldehyde, then excisedfrom the membrane insert (Millicell-CM, 0.4µm, Millipore). The cultures with membraneattached were processed free-floating, mak-ing sure that the membrane was facing thebottom at all times. The permeabilization stepwas an overnight incubation in 1% Triton X-100 in phosphate-buffered saline (PBS) at 4°C.The sections were then blocked in 20% bovineserum albumin with 0.1% TritonX-100 in PBSfor one hour at room temperature.

The primary antibodies used were glial fib-rillary acidic protein (GFAP, Abcam, ab4674,1:50), microtubule-associated protein 2,(MAP2, Chemicon, MAB3418, 1:200) and con-nexin-43 (Sigma, C6219, 1:200) made up in 1%normal goat serum with 0.1% TritonX-100 inPBS. All three antibodies were simultaneouslyapplied. Primary antibody incubation wasovernight at 4°C followed by incubation inspecies-appropriate secondary antibody forthree hours at room temperature. Formicroglia labelling, slices were incubated inAlexa 488-conjugated isolectin-�4 (Invitrogen,I-21411, 1:100) in PBS overnight at 4°C.

Bridge MountingOwing to the thickness of the cultured slices, abridge mounting technique was used as fol-lows: a 22 mm � 22 mm No.1 (170 µm-thick)

Improved Immunohistochemical Protocolfor Organotypic Brain Slice CulturesJinny J. Yoon,1 Vithika Suri,2 Louise F. B. Nicholson2 and Colin R. Green1

1. Department of Ophthalmology, and 2. Department of Anatomy with Radiology and the Centre for BrainResearch, University of Auckland, New Zealand.

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cryostat sections cut in the coronal (Figure 1d)and transverse (Figure 1e) planes. The trans-verse and cross-sections of the cultured slicesshowed that cells were present throughoutthe slice and the ‘blank space’ resulted from alabelling artifact. Sequential application ofmultiple antibodies one after the other didnot change the patterns of immunostaining,ruling out the possibility of steric hindrance ofantibody penetration (not shown).

Subsequently, varying the amounts of TritonX-100, a permeabilization agent, and differ-ent durations of incubation were tested. The‘blank space’ was observed without a perme-abilization step (Figure 2a). Triton X-100 at 1%for 15 minutes and 0.5 % overnight bothimproved antibody penetration but signalswere still weak (Figure 2b and 2c). Figure 2dand 2e show that overnight incubation in 1%Triton X-100 vastly improved antibody pene-tration and provided clear immunolabellingthrough the entire thickness of the slice.

D I SCUSS IONImmunohistochemical techniques previouslydescribed for whole mount brain slice culturesdo not fully take into account the thickness ofthe tissue when preparing the specimens formicroscopical examination.

Conventional coverslip mounting squashesslices down to less than one third of the thick-ness maintained with the bridge mountingmethod used here. This will significantly affect3D relationships within the tissue. A conven-tional No. 1 coverslip is typically 170 µm thickand in our case cultured slices (imagedthrough about 100 µm) were processedattached to the 50 µm-thick membrane. It ispossible to stack coverslips at either end toincrease the depth of the specimen well forthicker slices but two factors are important toconsider. First, image quality falls away veryquickly if there is gap between the tissue andthe coverslip, even if it is filled with mountingmedium. Second, the working distance will beconstrained by both the objective lens usedand the tissue transparency. In the case of theCNS, however, the tissue is relatively transpar-

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Figure 1: Problems associated with conventional immunohistochemical labelling of whole mount hippocampal slice cultures. Slices cultured for 12 days werelabelled with isolectin-�4 using conventional techniques and Z-series scans were taken using a confocal microscope (a, b and c). Slices were mountedusing coverslip bridges to avoid squashing. Representative images of top (a), middle at 50 µm from the surface (b) and bottom (c) of a whole mountslice are shown. Although labelling is clear near the top and bottom, there appears to be an unlabelled ‘blank space’ in the middle of the slice. The‘blank space’ cannot be identified in serial coronal cryostat sections (d) or transverse cryostat section cut perpendicular to the membrane (e). The sec-tion shown in (d) corresponds to 50 µm below the top in the CA3 region of a whole mounted slice. Red is MAP2 and green GFAP. The section in (e)shows the CA1 pyramidal cell payer. Red is MAP2, green connexin-43 and blue GFAP. Scale bars: a-c, 30 µm; d-e, 50 µm.

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ent enabling confocal imaging through atleast 100 µm with clarity.

The other issues that we have addressed areantibody labelling, penetration and mountingprotocols. Gogolla et al. [3] provide excellentprotocols for immunohistochemical labellingof tissue slices. The key differences here arethe comparison between cryosection of thesame tissue and whole mounts to determinean optimum permeabilization protocol andthe need to allow adequate time for antifademedium to fully penetrate before imaging.This ensures that antifade protection is main-tained through the entire slice thickness, andthat a homogeneous refractive index isobtained throughout the specimen.

CONCLUS IONSIn summary, the optimal steps for wholemount immunohistochemistry involved thebridge mounting technique to avoid slice cul-tures from being squashed and overnight incu-bation in 1% Triton X-100 which enhancedantibody penetration. The protocol estab-lished here has produced continuous tripleimmunolabelling through the full thickness ofcultured slices and represents an improvementfor investigating cellular morphology and 3Drelationships within slice culture models.

REFERENCES1. Noraberg, J. et al. Organotypic hippocampal slice cultures

for studies of brain damage, neuroprotection andneurorepair. Current Drug Targets-CNS and NeurologicalDisorders 4(4):435-452, 2005.

2. Sundstrom, L. et al. Organotypic cultures as tools forfunctional screening in the CNS. Drug Discovery Today10(14):993-1000, 2005.

3. Gogolla, N. et al. Staining protocol for organotypichippocampal slice cultures. Nature Protocols 1(5):2452-2456,2006.

4. Noraberg, J. et al. Markers for neuronal degeneration inorganotypic slice cultures. Brain Research: Brain ResearchProtocols 3(3):278-290, 1999.

5. Stoppini, L. et al. A simple method for organotypic culturesof nervous tissue. J. Neuroscience Methods 37(2):173-182,1991.

©2011 John Wiley & Sons, Ltd

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Figure 2: Comparison of penetration strategies in whole mount immunohistochemistry. Slices cultured for 12 days were processed for MAP2 immunohisto-chemistry as whole mounts. Confocal images were taken from the CA3 area and at about 50 µm from the surface. (a) Without using a penetrant. (b)1% Triton X-100 for 15 minutes. (c) Overnight in 0.5% Triton X-100. (d) Overnight incubation in 1% Triton X-100 provided markedly enhanced anti-body penetration resulting in consistent label throughout the entire thickness of the slice cultures. (e) The slice was triple labelled with GFAP (blue),MAP2 (red) and connexin-43 (green) after overnight incubation in 1% Triton X-100. The image was taken from the CA3 pyramidal cell layer and fromthe middle of a 100 µm-thick slice. The cellular morphology and laminar structure of the hippocampus are well preserved and comparable to that insitu. Scale bars: a-d, 50 µm; e, 20 µm.

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