Examples of fluorochromes that fit in the FDMM setup.

<p>* fluorochromes that were tested in the FDMM setup</p><p>Examples of fluorochromes that fit in the FDMM setup.</p

Six channel FDMM for subcellular staining of cultured NIH cells.

<p>Cultured NIH cells immunolabeled for different intracellular structures. Upper small pictures: Nuclei (DAPI channel, DAPI, 12 ms), Focal adhesions (425 channel, anti-vinculin, 90 ms), Lipid droplets (488 channel, anti-ADRP, 50 ms), Golgi (Cy3 channel, anti-syntaxin 5, 10 ms), F-actin cytoskeleton (594 channel, phalloidin, 17 ms), and Lyzosomes (PerCP channel, anti-LAMP 1, 46 ms). Scale bar, 40 μm. Objective x63/1.4 Oil DIC. Lower large picture: Merged image of all channels. Scale bar, 20 μm.</p

Detection of regulatory T cells in germinal centers using FDMM.

<p>Mice were immunized i.p. with a mixture of polyI:C and ovalbumin to induce formation of germinal centers. Ten days later the spleens were harvested, sectioned and multi-immunolabeled with antibodies against IgD-expressing B cells (anti-IgD, red), follicular dendritic cells (anti-MFG-E8, cyan), CD4⁺ T cells (anti-CD4, green), the transcription factor Foxp3 (anti-Foxp3, white), B cells (anti-B220, not shown) and nuclei (DAPI, blue). <i>(A)</i> Detection of a germinal center. The picture shows the expression of IgD (red, negative marker) and MGF-E8 (cyan, positive marker). <i>(B)</i> Detection of regulatory T cells (CD4⁺Foxp3⁺) in a germinal center (arrow) and outside a germinal center (arrowhead). All channels are presented individually in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119499#pone.0119499.s004" target="_blank">S4 Fig</a>. Scale bar, 50 μm.</p

Filter-Dense Multicolor Microscopy

<div><p>Immunofluorescence microscopy is a unique method to reveal the spatial location of proteins in tissues and cells. By combining antibodies that are labeled with different fluorochromes, the location of several proteins can simultaneously be visualized in one sample. However, because of the risk of bleed-through signals between fluorochromes, standard multicolor microscopy is restricted to a maximum of four fluorescence channels, including one for nuclei staining. This is not always enough to address common scientific questions. In particular, the use of a rapidly increasing number of marker proteins to classify functionally distinct cell populations and diseased tissues emphasizes the need for more complex multistainings. Hence, multicolor microscopy should ideally offer more channels to meet the current needs in biomedical science. Here we present an enhanced multi-fluorescence setup, which we call Filter-Dense Multicolor Microscopy (FDMM). FDMM is based on condensed filter sets that are more specific for each fluorochrome and allow a more economic use of the light spectrum. FDMM allows at least six independent fluorescence channels and can be applied to any standard fluorescence microscope without changing any operative procedures for the user. In the present study, we demonstrate an FDMM setup of six channels that includes the most commonly used fluorochromes for histology. We show that the FDMM setup is specific and robust, and we apply the technique on typical biological questions that require more than four fluorescence microscope channels.</p></div

The strategy and specificity of FDMM.

<p><i>(A)</i> Example of a condensed filter set compared with a standard filter set (Carl Zeiss, filter set #38) for the 488 channel. The curves show the excitation (blue line) and emission (red line) light spectra of the fluorochrome AF488. Blue rectangles, excitation filter interval; red rectangles, emission filter interval; vertical black line, beam splitter. <i>(B)</i> By the use of condensed filter sets, the density of fluorescence channels is increased in the FDMM setup compared with standard multicolor microscopy. <i>(C)</i> Each filter set in the FDMM setup is specific for its corresponding fluorochrome. Six tissue sections from a blood vessel were immunolabeled for CD31 (endothelium) with different fluorochromes or nuclei stained with DAPI. Each filter set (columns) received signals from only one fluorochrome (rows). Scale bar, 100 μm. Exposure times: DAPI channel (11 ms), 425 channel (30 ms), 488 channel (63 ms), Cy3 channel (20 ms), 594 channel (54 ms), PerCP channel (17 ms). Objective: x20/0.75.</p

Multicolor antibody array for mouse spleen using FDMM.

<p>Tissue sections from a wild type spleen (WT), a TNFα-receptor 1 knockout spleen (TNFaR1 KO), and a spleen from a CD11b-DTR mouse (CD11b-DTR) were multi-immunolabeled with antibodies against marginal zone macrophages (anti-CD169, cyan), dendritic cells (anti-CD11c, blue), B cells (anti-B220, red), CD4⁺ T cells (anti-CD4, green), and CD8⁺ T cells (anti-CD8, magenta). Nuclei were stained with DAPI (not shown). Arrow in TNFα-receptor 1 knockout spleen indicates weak marginal zone structure (cyan). Arrowhead in CD11b-DTR spleen indicates CD4⁺ T cells (green) within the B cell follicles. All six channels are presented individually in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119499#pone.0119499.s006" target="_blank">S6 Fig</a>. Scale bar, 100 μm.</p

Detection of lipid-loaded dendritic cells in an atherosclerotic lesion using FDMM.

<p>Atherosclerotic lesions were induced in the carotid artery of LDLr^−/− mice. The carotid arteries were harvested, sectioned and multi-immunolabeled with antibodies against lipid droplets (anti-perilipin 2, red), smooth muscle cells (anti-SM α-actin, cyan), antigen presenting cells (anti-MHC class II, green, upper left), CD11c (anti-CD11c, green, lower left), and CD11b (anti-CD11b, green, lower right). Nuclei were stained with DAPI (blue). Arrows depict cells that are positive for MHC class II, CD11c and perilipin 2, and negative/dim for CD11b. The small arrowheads indicate arterial elastin fibers (laminae), which autofluoresce in the 425 channel. All six channels are presented individually in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119499#pone.0119499.s005" target="_blank">S5 Fig</a>. Scale bar, 100 μm.</p

ARF6-dependent neuronal differentiation is normalized after inhibition of glucosylceramide synthase.

<p>(<b>A</b>) Quantification of long outgrowth from Neuro-2a cells transfected with control or ARF6 siRNA in medium with 10% FCS for 48 h and then differentiated for 24 h in medium without serum in the absence or presence of D-PDMP (10 µmol/l). <i>n</i> = 4 per group, ***<i>P</i><0.001 <i>vs</i> control; †††<i>P</i><0.001 <i>vs</i> absence of D-PDMP. (<b>B</b>) Cellular levels of ceramides (Cer), sphingomyelin (SM) and glucosylceramide (GC). <i>n</i> = 4–5 per group, **<i>P</i><0.01 <i>vs</i> control, ***<i>P</i><0.001 <i>vs</i> control.</p

ARF6 knockdown increases glucosylceramide levels and decreases sphingomyelin levels in Neuro-2a cells.

<p>Neuro-2a cells were transfected with control (c) or ARF6 siRNA in medium with 10% FCS and lipids were extracted after 48 h. (<b>A</b>) Cellular levels of ceramides (Cer), sphingomyelin (SM) and glucosylceramide (GC) and (<b>B</b>) phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) were analyzed as described in the methods section. <i>n</i> = 4 per group, **<i>P</i><0.01, ***<i>P</i><0.001 <i>vs</i> control.</p

ARF6 knockdown increases cellular glucosylceramide levels through increased glucosylceramide synthase activity in Neuro-2a cells.

<p>(<b>A</b>) Schematic overview of the sphingolipid synthesis pathway. (<b>B–D</b>) Neuro-2a cells were transfected with control (c) or ARF6 siRNA in medium with 10% FCS and analyzed after 48 h. (<b>B</b>) mRNA expression of glucosylceramide synthase (GCS) in RNA extracted from Neuro-2a cells (<i>n</i> = 6 per group). (<b>C</b>) Representative image of a TLC plate from a GCS activity assay showing increased levels of synthesized NBD-glucosylceramide in Neuro-2a cells transfected with ARF6 siRNA. (<b>D</b>) Quantification of increased glucosylceramide synthase activity after ARF6 knockdown (<i>n</i> = 4 per group). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 <i>vs</i> control.</p