The authors thank S. Tausch for excellent technical assistance. The work was supported by BMBF (Bernstein Focus 01GQ0923) and DFG (FOR1738).

NOTE: All procedures involving living animals were carried out in accordance with the EC directive 86/609/EEC guidelines on the care and use of laboratory animals and approved by the local ethics committee (Th&#252;ringer Landesamt f&#252;r Lebensmittelsicherheit und Verbraucherschutz).
1. Intraperitoneal Injection of Thymidine Analogs
<ol>
	<li>Weigh animals the day before injection. Calculate the amount of thymidine analog required for all injections planned on the next day as well as indi...

<ol>
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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+doses+of+bromo-+and+iododeoxyuridine+produce+near-saturation+labeling+of+adult+proliferative+populations+in+the+dentate+gyrus.">Low doses of bromo- and iododeoxyuridine produce near-saturation labeling of adult proliferative populations in the dentate gyrus.</a> Eur J Neurosci. 21 (3), 803-807 (2005).</li><li>Cameron, H. A., McKay, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+neurogenesis+produces+a+large+pool+of+new+granule+cells+in+the+dentate+gyrus.">Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus.</a> J Comp Neurol. 435 (4), 406-417 (2001).</li><li>Vega, C. J., Peterson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+proliferative+history+in+tissue+revealed+by+temporal+halogenated+thymidine+analog+discrimination.">Stem cell proliferative history in tissue revealed by temporal halogenated thymidine analog discrimination.</a> Nat Methods. 2 (3), 167-169 (2005).</li><li>Scholzen, T., Gerdes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ki-67+protein:+from+the+known+and+the+unknown.">The Ki-67 protein: from the known and the unknown.</a> J Cell Physiol. 182 (3), 311-322 (2000).</li><li>Brown, D. C., Gatter, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ki67+protein:+the+immaculate+deception.">Ki67 protein: the immaculate deception.</a> Histopathology. 40 (1), 2-11 (2002).</li><li>Yamaguchi, M., Saito, H., Suzuki, M., Mori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+neurogenesis+in+the+central+nervous+system+using+nestin+promoter-GFP+transgenic+mice.">Visualization of neurogenesis in the central nervous system using nestin promoter-GFP transgenic mice.</a> Neuroreport. 11 (9), 1991-1996 (2000).</li><li>Kempermann, G., Kuhn, H. G., Gage, F. 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Quantification and identification of subpopulations of newborn cells is a central issue in adult neurogenesis research. Combining proliferation markers and antibodies against proteins expressed during specific stages of adult neurogenesis allows immunohistochemical detection of these subpopulations. Some of the antibodies or antibody combinations require specific staining conditions.
Labeling of dividing cells with synthetic thymidine analogs is still the gold standard for studying adult hippo...

Two brain regions constitutively generate new neurons throughout life, the subventricular zone of the lateral ventricles and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG). The newborn neurons derive from neural progenitor cells and go through different stages of morphological and physiological development before reaching maturity1,2. From a slowly dividing radial glia-like stem cell (type 1) consecutive stages of transit amplifying intermediate progenitor cells arise. The more undifferentiated subtypes (type 2a and type 2b) have an irregular shape with short, tangential processes. They generate neuroblasts (type 3) that gradually exit the cell cycle to become immature neurons (with dendrites extended towards the molecular layer) and finally integrate into the hippocampal network as mature granule cells. Due to their particular physiological characteristics these cells provide the circuitry with enhanced plasticity3 suggesting a unique role in hippocampal function. Actually, studies of the last decade generated substantial evidence that adult neurogenesis contributes to spatial memory, pattern separation and emotional behavior4,5.
Adult neurogenesis can be studied using different approaches. Thymidine analogs incorporate into DNA during the S-phase of the cell cycle and allow birth dating, quantification and fate analysis of newborn cells6-8. Sequential application of different thymidine analogs (e.g., CldU, EdU or IdU) can be used to study cell turnover or cell populations born at different time points during the course of an experiment9. An alternative, endogenous marker for cell proliferation is Ki67. It is expressed in dividing cells during all phases of the cell cycle (G1, S, G2, M) except the resting phase (G0) and the beginning of G1&#160;10,11. To analyze the phenotype of newborn cell populations in the adult dentate gyrus several stage-specific molecular markers can be used such as GFAP, nestin, DCX and NeuN1,6. GFAP is a marker of mature astrocytes but is also expressed in radial glia-like cells in the adult forebrain. Nestin is an intermediate filament specific for radial glia-like cells and early intermediate progenitor cells. DCX is a microtubule-associated protein expressed in intermediate progenitors, neuroblasts and immature neurons. Based on the (co-) expression of these three markers and the morphological features of the labeled cells four distinct progenitor cell subtypes can be identified: type 1 (GFAP+, nestin+, DCX-), type 2a (GFAP-, nestin+, DCX-), type 2b (GFAP-, nestin+, DCX+) and type 3 (GFAP-,nestin-, DCX+)1. Co-labeling of DCX together with NeuN, which is expressed in postmitotic neurons, allows the differentiation of immature (DCX+, NeuN+) and mature (DCX-, NeuN+) granule neurons.
The above mentioned markers are frequently used for immunofluorescent co-labeling and subsequent confocal microscopy to analyze the number and identity of newborn cells. This typically requires antibodies from different host species to prevent undesired antibody cross-reactivity. However, the majority of primary antibodies suitable for neurogenesis research are raised either in rabbits or mice (e.g., mouse &#945;-BrdU, mouse &#945;-NeuN, rabbit &#945;-Ki67, rabbit &#945;-GFAP). This leads to serious limitations in the number and combination of antigens that could be evaluated in a single slice. This in turn not only increases the staining effort, as multiple stainings have to be performed, but might also compromise the reliability of results. Furthermore, some antigens are susceptible to formalin fixation-induced epitope masking (e.g., Ki67, nestin). We herein describe modifications from the classical single- and multiple immunolabeling protocols (e.g., epitope retrieval, multiple sequential immunostaining, use of nestin-GFP transgenic mice12) that overcome many of these issues. In particular, the sequential multiple immunofluorescence protocol allows staining against up to four different antigens even if part of the antibodies is derived from the same host. This enables the simultaneous detection of type 1, type 2a, type 2b and type 3 progenitor cells, as well as their proliferative activity within a single section.

<table border="0" cellpadding="0" cellspacing="0" width="665">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:277px;">Name</td>
			<td style="width:147px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:131px;">Comments &#38; Dilutions</td>
		</tr>
		<tr height="13">
			<td height="13" style="height:13px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thymidine analog administration</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-Bromo-2&#8242;-deoxyuridine, BrdU</td>
			<td>Sigma-Aldrich</td>
			<td>B9285</td>
			<td rowspan="4" style="width:131px;">toxic (mutagenic, teratogenic)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-Chloro-2&#8242;-deoxyuridine, CldU</td>
			<td>Sigma-Aldrich</td>
			<td>C6891</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-Chloro-2&#8242;-deoxyuridine, CldU</td>
			<td>MP Biomedicals</td>
			<td>2105478</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-Iodo-2&#8242;-deoxyuridine, IdU</td>
			<td>MP Biomedicals</td>
			<td>2100357</td>
		</tr>
		<tr height="10">
			<td height="10" style="height:11px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane-Actavis</td>
			<td>Piramal Healthcare</td>
			<td>700211</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Paraformaldehyde powder (PFA)</td>
			<td>Riedel-De H&#228;en</td>
			<td>16005</td>
			<td>toxic, flammable</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Perfusion pump PD5206</td>
			<td>Heidolph Instruments</td>
			<td>523-52060-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Masterflex Tygon lab tubing, &#216; 0.8 mm&#160;</td>
			<td>Thermo Fischer Scientific</td>
			<td>06409-13</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Feeding needle, straight, 21G, 1.75mm olive tip, 40mm</td>
			<td>Agnthos</td>
			<td>1036</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Freezing microtome Microm HM 400</td>
			<td>Thermo Fischer Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">24 Well Cell Culture Multiwell Plates</td>
			<td style="width:147px;">Greiner Bio-One</td>
			<td>662160</td>
			<td></td>
		</tr>
		<tr height="10">
			<td height="10" style="height:11px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunohistochemistry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tefal Vitacuisine Steamer</td>
			<td>Tefal</td>
			<td>VS 4001</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:277px;">Netwell 24mm Polyester Mesh Membrane Inserts Pre-Loaded in 6-Well Culture Plates</td>
			<td>Corning</td>
			<td>3479</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:277px;">Netwell 15mm Polyester Mesh Membrane Inserts Pre-Loaded in 12-Well Culture Plates</td>
			<td>Corning</td>
			<td>3477</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:277px;">Netwell Plastic 6-Well Carrier Kit for 24mm Polyester Mesh Membrane Inserts</td>
			<td>Corning</td>
			<td>3521</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:277px;">Netwell Plastic 12-Well Carrier Kit for 15mm Polyester Mesh Membrane Inserts</td>
			<td>Corning</td>
			<td>3520</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vectastain Elite ABC Kit</td>
			<td>Vector Laboratories</td>
			<td>PK-6100</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:277px;">DAB (3,3&#8242;-Diaminobenzidine tetrahydrochloride hydrate)</td>
			<td>Sigma-Aldrich</td>
			<td>D-5637</td>
			<td style="width:131px;">carcinogenic, light sensitive</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Fluoromount-G</td>
			<td>SouthernBiotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;">rabbit IgG1 &#945;-Ki67</td>
			<td style="width:147px;">Novocastra/ Leica Biosystems</td>
			<td>NCL-L-Ki67MM1</td>
			<td style="width:131px;">DAB 1:400/IF 1:100; requires epitope retrieval</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rabbit &#945;-GFAP, AS-3-GF</td>
			<td>Synaptic Systems</td>
			<td>173 002</td>
			<td>1:500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">goat IgG (H+L) &#945;-GFP</td>
			<td>Acris Antibodies</td>
			<td>R1091P</td>
			<td>1:300</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">mouse IgG1 &#945;-nestin</td>
			<td>Abcam</td>
			<td>ab6142</td>
			<td style="width:131px;">1:200; requires epitope retrieval</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">guinea pig IgG (H+L) &#945;-Doublecortin</td>
			<td>Merck Millipore</td>
			<td>AB2253</td>
			<td>1:500</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">rat IgG2a &#945;-BrdU (ascites)</td>
			<td>AbD Serotec/ Bio-Rad</td>
			<td>OBT0030CX</td>
			<td style="width:131px;">for detection of BrdU; DAB 1:500/IF 1:400</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">rat IgG2a &#945;-BrdU (purified)</td>
			<td>AbD Serotec/ Bio-Rad</td>
			<td>OBT0030&#160;&#160;</td>
			<td style="width:131px;">for detection of CldU; DAB 1:500/IF 1:250-400</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">mouse IgG1&#312; &#945;-BrdU</td>
			<td>BD Biosciences</td>
			<td>347580</td>
			<td style="width:131px;">for detection of IdU; DAB 1:500/IF 1:350</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mouse IgG1 &#945;-NeuN</td>
			<td>Merck Millipore</td>
			<td>MAB377</td>
			<td>1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">donkey &#945;-guinea pig IgG (H+L)-Biotin</td>
			<td>Dianova</td>
			<td>711-065-152</td>
			<td>1:500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">donkey &#945;-rat IgG (H+L)-Biotin</td>
			<td>Dianova</td>
			<td>712-065-150</td>
			<td>1:500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">donkey &#945;-mouse IgG (H+L)-Biotin</td>
			<td>Dianova</td>
			<td>715-065-151</td>
			<td>1:500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">goat &#945;-rat IgG (H+L)-Alexa Fluor 488</td>
			<td>Molecular Probes</td>
			<td>A11006</td>
			<td>1:250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">donkey &#945;-goat IgG (H+L)-Alexa Fluor 488</td>
			<td>Molecular Probes</td>
			<td>A11055</td>
			<td>1:250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">donkey &#945;-mouse IgG (H+L)-FITC, Fab-Fragment</td>
			<td>Dianova</td>
			<td>715-097-003</td>
			<td>1:100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">donkey &#945;-mouse IgG (H+L)-Alexa Fluor 647</td>
			<td>Dianova</td>
			<td>715-605-151</td>
			<td>1:250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">donkey &#945;-guinea pig IgG (H+L)-Alexa Fluor 647</td>
			<td>Dianova</td>
			<td>706-605-148</td>
			<td>1:250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">donkey &#945;-rat IgG (H+L)-Rhodamine Red-X</td>
			<td>Dianova</td>
			<td>712-295-150</td>
			<td>1:250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">donkey &#945;-rabbit IgG (H+L)-Rhodamine Red-X</td>
			<td>Dianova</td>
			<td>711-295-152</td>
			<td>1:250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">donkey &#945;-guinea pig IgG (H+L)-Rhodamine Red-X</td>
			<td>Dianova</td>
			<td>706-296-148</td>
			<td>1:250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptavidin-Rhodamine Red-X&#160;</td>
			<td>Dianova</td>
			<td>016-290-084</td>
			<td>1:500</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">goat &#945;-rabbit IgG (H+L)-AMCA</td>
			<td>Dianova</td>
			<td>111-155-144</td>
			<td style="width:131px;">1:250, works only with rabbit &#945;-GFAP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hoechst 33342</td>
			<td>Molecular Probes</td>
			<td>H3570</td>
			<td>1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Molecular Probes</td>
			<td>D1306</td>
			<td>1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blocking</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fab-fragment donkey &#945;-mouse IgG (H+L)</td>
			<td>Dianova</td>
			<td>715-007-003</td>
			<td>1:20</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fab-fragment donkey &#945;-rabbit IgG (H+L)</td>
			<td>Dianova</td>
			<td>711-007-003</td>
			<td>1:20</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Normal donkey serum</td>
			<td>Merck Millipore</td>
			<td>S30</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Normal rabbit serum</td>
			<td>Dianova</td>
			<td>011-000-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Normal goat serum</td>
			<td>Dianova</td>
			<td>005-000-001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Serum Albumine</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Histology</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Cresyl violett</td>
			<td>Sigma-Aldrich</td>
			<td>C5042</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:277px;">Neo-Clear</td>
			<td>Merck Millipore</td>
			<td>109843</td>
			<td style="width:131px;">non-toxic xylene substitute</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:277px;">Neo-Mount</td>
			<td>Merck Millipore</td>
			<td>109016</td>
			<td style="width:131px;">permanent mounting medium</td>
		</tr>
		<tr height="10">
			<td height="10" style="height:11px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscopy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Axioskop 2</td>
			<td>Carl Zeiss Microscopy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">LSM 710</td>
			<td>Carl Zeiss Microscopy</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

immunohistochemistry and multiple labeling with antibodies from the same host species to study adult hippocampal neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present&#160;a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

We applied the methods described above to quantify and characterize newborn cells in the postnatal and adult hippocampus. Therefore, we used wildtype and neurogenesis-deficient cyclin D2 knock out (Ccnd2KO) mice housed under conditions known to affect the rate of neurogenesis (i.e., enriched environment, EE)13,14. Immunohistochemical DAB staining against either Ki67, BrdU, CldU or IdU consistently revealed differences in newborn cell numbers between wildtype and Ccnd2KO mice (<strong...

Watch this Scientific Journal Video about Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis at JoVE.com

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

immunohistochemistry-multiple-labeling-with-antibodies-from-same-host

Research

JoVE Journal

Neuroscience

26.4K vistas.  Jena University Hospital. This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.