Histochem Cell Biol (2008) 130:157–164 DOI 10.1007/s00418-008-0417-8
ORIGINAL PAPER
Immunohistologic techniques for detecting the glycolipid Gb3 in the mouse kidney and nervous system Glynis L. Kolling · Fumiko Obata · Lisa K. Gross · Tom G. Obrig
Accepted: 11 March 2008 / Published online: 26 March 2008 © Springer-Verlag 2008
Abstract Shiga toxin-producing Escherichia coli causes hemolytic uremic syndrome, a constellation of disorders that includes kidney failure and central nervous system dysfunction. Shiga toxin binds the amphipathic, membranebound glycolipid globotriaosylceramide (Gb3) and uses it to enter host cells and ultimately cause cell death. Thus, cell types that express Gb3 in target tissues should be recognized. The objective of this study was to determine whether immunohistologic detection of Gb3 was aVected by the method of tissue preparation. Tissue preparation included variations in Wxation (immersion or perfusion) and processing (paraYn or frozen) steps; paraYn processing employed diVerent dehydration solvents (acetone or ethanol). Perfusion-Wxation in combination with frozen sections or acetone-dehydrated tissue for paraYn sections resulted in speciWc recognition of Gb3 using immunohistochemical or immunoXuorescent methods. In the mouse tissues studied, Gb3 was associated with tubules in the kidney and neurons in the nervous system. On the other hand, Gb3 localization to endothelial cells was determined to be an artifact generated due to immersion-Wxation or tissue dehydration with ethanol. This Wnding was corroborated by glycolipid proWles from tissue subjected to dehydration; namely Gb3 was subject to extraction by ethanol more than acetone during tissue dehydration. The results of this study show that tissue preparation is crucial to the persistence and
G. L. Kolling and F. Obata contributed equally to this work. G. L. Kolling · F. Obata · L. K. Gross · T. G. Obrig (&) Department of Internal Medicine/Nephrology, University of Virginia, Box 800133, Charlottesville, VA 22908, USA e-mail:
[email protected]
preservation of the glycolipid Gb3 in mouse tissue. These methods may serve as a basis for determining the localization of other amphipathic glycolipids in tissue. Keywords Globotriaosylceramide (Gb3) · Hemolytic uremic syndrome · Shiga toxin
Introduction Infection with Escherichia coli O157:H7 or other Shiga toxin-producing E. coli can cause kidney failure and dysfunction of multiple organ systems, including the central nervous system (CNS) (Taylor et al. 1999). Shiga toxin is a potent cytotoxin that binds to the glycolipid receptor globotriaosylceramide (Gb3). After binding to Gb3, Shiga toxin enters cells through receptor-mediated endocytosis and eventually disrupts protein synthesis, resulting in cell dysfunction and death. Therefore, identiWcation of cell types that express Gb3 in target tissues could provide information regarding disease pathogenesis and ultimately treatment. In vivo studies of humans and baboons show that renal Gb3 localizes to glomeruli and tubules (Chark et al. 2004; Clayton et al. 2005; Ergonul et al. 2003; Lingwood 1994). In the murine kidney, Gb3 is associated primarily with cortical and medullary tubules (Fujii et al. 2005; Okuda et al. 2006; Rutjes et al. 2002); however, at least one study contends that Gb3 is expressed on the renal endothelium (Okuda et al. 2006). In nervous system tissue, Gb3 is expressed by sensory neurons of the dorsal root ganglion in human, mouse, rabbit, and rat. Capillaries in human and rabbit nervous systems show Gb3, but those from mouse and rat do not (Ren et al. 1999). Conversely, a recent study showed endothelial Gb3 in the mouse brain (Okuda et al. 2006). To date,
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no consensus has been reached regarding the association of Gb3 with murine endothelial tissue. Tissues can be prepared for histologic analysis using several methods, but each has advantages and disadvantages. For studies of the amphipathic Shiga toxin receptor, frozen-section methods typically are used. However, even quick-freezing procedures may produce ice crystals that damage membranes. Alternatively, tissues can be embedded in paraYn, which results in superior preservation of morphology when compared with quick-frozen samples, but the ethanol used for tissue dehydration may extract lipids (Elleder and Lojda 1973). In this study, we examined several methods of tissue preparation to determine their ability to preserve Gb3 in the murine kidney and nervous system. We describe a method of paraYn embedding that retains Gb3 and detail a more sensitive frozen-section method that may be used to examine cellular Gb3 localization.
Materials and methods Animals Normal, male, C57BL/6 mice (mean mass, 22–24 g) were purchased from Charles River Laboratories (Wilmington, Massachusetts). Food and water were provided ad libitum. Animals were euthanized by asphyxiation with carbon dioxide. All animal experiments were conducted according to protocols approved by the Animal Care and Use Committee at the University of Virginia (Charlottesville, Virginia). Tissue Wxation Perfusion-Wxation (using a peristaltic pump) was performed by perfusing saline through the left cardiac ventricle and following with 4% paraformaldehyde in phosphatebuVered saline (PFA/PBS). Dissected tissues were further Wxed in 4% PFA/PBS for up to 4 h at 4°C. Immersion-Wxation was performed by immersing dissected tissue in 4% PFA/PBS for 4 h at 4°C (no perfusion). For paraYnembedded preparations, the total Wxation time was extended to 24 h. Tissue dehydration for paraYn embedding Perfusion- or immersion-Wxed tissues were dehydrated using ethanol or acetone. The standard dehydration procedure used an ethanol gradient at 40°C: 70% ethanol for 10 min (1£); 95% ethanol for 5 min (1£); 95% ethanol for 10 min (2£); 100% ethanol for 10 min (2£); and 100% ethanol for 5 min (1£). After dehydration, samples were
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immersed in xylene (5 min [1£]; 10 min [1£]) and inWltrated with paraYn. The modiWed dehydration procedure used an acetone gradient at 4°C: 25% acetone for 20 min (2£); 50% acetone for 20 min (2£); 75% acetone for 45 min (2£); 95% acetone for 45 min (2£); and 100% acetone for 45 min (2£). Samples were immersed in roomtemperature xylene (10 min [2£]) and inWltrated with paraYn. After paraYn inWltration, tissues were embedded in paraYn blocks. Immunohistochemical detection of Gb3 in paraYn-embedded tissue ParaYn sections (slice thickness, 3 m) were placed on positively charged slides and incubated overnight at 37°C. Sections were deparaYnized using xylene and rehydrated with an ethanol or acetone gradient (reversal of the dehydration procedure described above). Sections were blocked to eliminate endogenous peroxidase activity and subjected to microwave antigen retrieval (using 10 mM citrate buVer, pH 6.0). The remaining steps were performed in a humidiWed chamber at room temperature. Sections were cooled after microwave antigen retrieval, endogenous biotin activity was blocked using an avidin/biotin blocking kit (VECTOR Laboratories, Burlingame, California), and endogenous IgM was blocked with goat anti-rat IgM (H&L) F(ab⬘)2, diluted 1:50 in goat sera. Antibodies used in this study are summarized in Table 1. Tissue sections were incubated with anti-Gb3/ CD77 monoclonal rat antibody or isotype-matched control rat IgM at room temperature for 1 h, washed with PBS, incubated for 30 min with biotinylated goat anti-rat IgM (H&L) F(ab⬘)2, and washed with PBS. The same staining procedures were used for all antibodies and matched isotype control antibodies. Immunoreactivity of the antibody–antigen complex was detected using Vectastain ELITE ABC reagent and the DAB Substrate kit (VECTOR Laboratories). Nuclei were visualized using hematoxylin as the counterstain. Stained slides were examined using a BX 40 microscope (Olympus, Center Valley, Pennsylvania). ImmunoXuorescent detection of Gb3 in Wxed, cryoprotected, free-Xoating tissue sections All incubations were performed at 4°C with gentle rocking. After washing in PBS, Wxed tissue samples were trimmed into smaller pieces and incubated overnight in phosphate-buVered, 30% sucrose. Sections were cut with a freezing microtome (slice thickness, 50 m), collected in phosphate-buVered, 30% sucrose, and stored at ¡80°C until use. Slices were blocked in U-shaped, 96-well plates for 1 h with anti-rat IgM antibody (for reactions
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Table 1 Antibodies used in this study Dilution ParaYn
Catalog No.
Clone No.
Company
Free-Xoating
Primary Ab, subtype Anti-Gb3/CD77, rat IgM
1:40
1:100
IM0175
38.13
Beckman Coulter
Anti-NeuN, mouse IgG1
1:1,000
1:1,000
MAB377
A60
Millipore/Chemicon International, Inc.
Anti-GFAPa, mouse IgG1
N.A.
1:1,000
C9205
G-A-5
Sigma-Aldrich
Anti-GFAP, rabbit IgG
1:25,000
N.A.
Z 0334
N.A.
DakoCytomation
Anti-mouse CD31, rat IgG2a
N.A.
1:50
550274
MEC13.3
BD Pharmingen
Rat IgM
1:25
1:100
CBL607
N.A.
Millipore/Chemicon International, Inc.
Goat IgG
1:5000
N.A.
S-1000b
N.A.
Vector Laboratories
Mouse IgG1
1:100
1:100
CBL600
N.A.
Millipore/Chemicon International, Inc.
Rabbit IgG
1:20,000
N.A.
PP64
N.A.
Millipore/Chemicon International, Inc.
Rat IgG2a
N.A.
1:1000
CBL605
N.A.
Millipore/Chemicon International, Inc.
1:500
N.A.
F104BN
N.A.
American Qualex
Anti-rat IgM, Alexa Fluor 488
N.A.
1:2,000
A-21212
N.A.
Invitrogen/Molecular Probe
Anti-goat IgG, biotin
N.A.c
N.A.
PK-6105
N.A.
Vector Laboratories
Anti-mouse IgG, biotin
N.A.d
N.A.
PK-6102
N.A.
Vector Laboratories
Anti-mouse IgG, Alexa Fluor 546
N.A.
1:2,000
A-11060
N.A.
Invitrogen/Molecular Probe
Anti-rat IgG, Alexa Fluor 647
N.A.
1:2,000
A-21247
N.A.
Invitrogen/Molecular Probe
N.A.
PK-6101
N.A.
Vector Laboratories
Isotype-matched Ig
Secondary Ab, Probe Anti-rat IgM, biotin
Anti-rabbit IgG, biotin
e
N.A.
Abbreviations: Ab, antibody; Ig, immunoglobulin; N.A., not applicable a Cy3-conjugated primary Ab b Normal goat serum c Followed instructions from the VECTASTAIN Elite ABC kit (Goat IgG) d Followed instructions from the VECTASTAIN Elite ABC kit (Mouse IgG) e Followed instructions from the VECTASTAIN Elite ABC kit (Rabbit IgG)
containing anti-Gb 3/CD77 antibody or its isotype control rat IgM) or with secondary antibody-matched normal sera (1:20 dilution). After blocking, slices were incubated with a primary antibody or isotype control antibody; incubations lasted 2 h, except when using antiGb3/CD77 antibody or rat IgM (both had overnight incubations). Samples were washed in PBS for 1 h, and samples were incubated with Xuorescent secondary antibodies for 2 h. Cy3-conjugated anti-glial Wbrillary acidic protein (GFAP) antibody did not require a Xuorescent secondary antibody. Samples were washed for 1 h and stained with 4,6-diamidino-2-phenylindole-2HCl (DAPI) (Molecular Probes/Invitrogen; Carlsbad, California) to visualize nuclei. Slices were placed in mounting media (GEL/MOUNT, Biomeda, Foster City, California) on glass slides and cover slipped. Specimens were examined with an LSM 510 microscope (Zeiss, Thornwood, NY, USA) equipped with a purple diode laser. Results were analyzed using LSM Image Browser software (version 4.0.0.157, Zeiss).
Lipid extraction and analysis with thin-layer chromatography and toxin overlay Mouse kidneys were dehydrated in ethanol or acetone and xylene as described above. Control kidneys did not receive any dehydration prior to lipid extraction. Lipids were extracted as previously described (Rose and Oklander 1965). BrieXy, half of 1 kidney was homogenized in 500 L of PBS, mixed with 2.2 mL of isopropanol, and vortexed intermittently for 1 h. Chloroform (1.4 mL) was added to samples and incubated with shaking for 1 h. Samples were Wltered through a glass wool plug in a Pasteur pipette and dried under nitrogen gas at 60°C. Samples were resuspended in chloroform/methanol (2:1) and loaded in duplicate on separate silica plates (Machery-Nagel, Bethlehem, Pennsylvania) along with authentic standards (neutral glycosphingolipid qualmix and Gb3, Matreya LLC, Pleasant Gap, Pennsylvania). Plates were developed in chloroform/methanol/water (65:25:4, v/v). Glycolipids on one plate were visualized using 8% CuSO4 · 5H2O in water/methanol/H3PO4 (60:32:8, v/v)
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Fig. 1 Gb3 localization in the murine kidney. a and b Immersion-Wxed tissue was dehydrated with acetone before paraYnization. Antibody-based detection of Gb3 using anti-Gb3/CD77 show tubular localization (a, arrows); nonspeciWc interactions are absent in tissue probed with isotype control antibodies (b). c and d In contrast, detection of Gb3 on tissue dehydrated with ethanol before paraYnization (c) or isotype control antibodies (d) show similar staining patterns in glomeruli (arrowheads) indicative of nonspeciWc antibody recognition. e–i In each frame, the white text box indicates the color corresponding to the speciWc reagent used (see Table 1) used for probing kidney sections by the free-Xoating method. e Gb3 is conWned to tubules (arrows) and not glomeruli (arrowheads) similar to the pattern seen in a. Increased magniWcation shows tubular localization of Gb3 (f) while glomeruli remain negative (h); nonspeciWc interations from the isotype control antibody (rat IgM) were absent in tubules (g) and glomeruli (i). Scale bars indicate 100 m (a–e) or 10 m (f–i)
(Abe et al. 2000). Gb3 on the duplicate plate was detected using Shiga toxin 1B (Stx1B) in a toxin overlay assay as previously described (Nutikka et al. 2003). BrieXy, the developed plate was blocked in Wsh gelatin [1% in TrisbuVered saline (TBS)], followed by incubation with Stx1B (0.2 g/mL). The plate was probed with an antiStx1B polyclonal rabbit antibody (diluted 1:1,000 in TBS). A horseradish peroxidase conjugated goat anti-rabbit secondary antibody (diluted 1:2,000 in TBS) was used to detect the lipid-toxin-antibody complex. Lipid spots
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were conWrmed by developing plates with 4-chloro1-naphthol.
Results IdentiWcation of Gb3 in murine kidney tubules ParaYn sections from immersion-Wxed tissue processed with diVerent dehydration solvents yielded diVerential
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IdentiWcation of Gb3 in murine neurons
Fig. 2 Thin-layer chromatography of neutral lipids from the murine kidney. Neutral lipid standards (Std) from bovine and porcine sources include glucosylceramide (GC), lactosylceramide (LC), globotriaosylceramide (Gb3), and globotetrosylceramide (Gb4). Neutral lipids were extracted from fresh mouse kidneys (Fresh), ethanol-dehydrated kidneys (EtOH), and acetone-dehydrated kidneys (Acet). The complete neutral lipid proWle (a) shows that ethanol dehydration eliminates substantially more lipids than acetone dehydration. b The Stx1B overlay method demonstrates that Gb3 levels are dramatically reduced by ethanol compared to acetone
antibody-based detection of Gb3 using anti-Gb3/CD77. In acetone-dehydrated kidney tissue, Gb3 localized with tubules (Fig. 1a) while isotype control antibodies were unreactive (Fig. 1b). In contrast, probing ethanol-dehydrated tissue with either anti-Gb3/CD77 (Fig. 1c) or isotype control antibodies identiWed similar patterns within glomeruli and between tubules (Fig. 1d). To validate Wndings from paraYn sections, antibodybased localization of Gb3 was carried out in frozen sections of perfusion-Wxed, cryoprotected tissue using the free-Xoating method. A tissue sample probed with anti-Gb3/CD77 antibodies, anti-CD31 antibodies (to identify glomerular endothelium), and stained with DAPI showed Gb3 in association with tubules only (Fig. 1e). Closer examination of tissues probed with anti-Gb3/CD77 or the isotype control antibodies conWrmed Gb3 localization to only the tubules (Fig. 1f, g) and not the glomerular endothelium (Fig. 1h, i). After kidneys were dehydrated with acetone or ethanol and xylene, lipids were extracted and separated by thinlayer chromatography (Fig. 2). Extracts from dehydrated kidneys (Fig. 2, “EtOH and Acet” lanes) were compared to kidneys that were not dehydrated (Fig. 2, “Fresh” lane). Regardless of the solvent used, dehydration decreased the total amount of extractable lipids (Fig. 2a). Importantly, Gb3 was retained during acetone treatment but ablated during ethanol dehydration (Fig. 2b).
Perfusion-Wxed, paraYn-embedded tissue from the murine nervous system was prepared in the same manner as that of the kidneys. Antibody-based detection of Gb3 using antiGb3/CD77 antibodies or the isotype control antibodies showed its association with neurons, regardless of the dehydration solvent used (Fig. 3). Figure 3a and b show tissue prepared with acetone, and Fig. 3c and d show tissue prepared with ethanol. Overall, the staining intensity of Gb3 was superior in acetone-dehydrated tissue compared to that in ethanol-dehydrated tissue. Antibody-based detection of free-Xoating sections was used to conWrm the cell type expressing Gb3 in perfusionWxed samples. The only cells containing Gb3 were large, nucleated cells identiWed as neurons because of their reactivity to the neuron-speciWc marker NeuN (Obata et al. 2008). In addition, Gb3 localization did not overlap with other cell types such as astrocytes (identiWed with antiGFAP antibodies) or endothelial cells (identiWed with antiCD31 antibodies) (Fig. 4). EVect of immersion-Wxation on Gb3 localization Kidney tissue that was Wxed by immersion rather than perfusion and used in the free-Xoating technique for antibodybased recognition of Gb3 resulted in localization to the tubular interstitium and glomeruli (Fig. 5a). However, on closer examination, Gb3-positive portions within glomeruli did not colocalize with the endothelial marker CD31 (Fig. 5b); moreover intravessel Gb3 localization also occurred in tissue stained with isotype control antibodies (Fig. 5c). Similarly, immersion-Wxed CNS tissue also showed colocalization of Gb3 and CD31 in free-Xoating sections (Fig. 6a). However, isotype control antibodies nonspeciWcally reacted with vessel-like structures (Fig. 6b) suggesting that antibody-based recognition of endothelial Gb3 was a false-positive. Similarly, immersion-Wxed, paraYnembedded nervous tissue sections showed false-positive staining of vessels (Fig. 6c) compared with isotype control antibodies (Fig. 6d).
Discussion We have described methods for successful tissue preservation and subsequent localization of physiologic Gb3 in the murine kidney and nervous system. Antibody-based detection of Gb3 in Wxed, paraYn-embedded tissue was aVected by the dehydration solvent. In free-Xoating sections, the Wxation method inXuenced accurate Gb3 detection. Gb3 recognition by anti-Gb3/CD77 was restricted to tubules in the
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Fig. 3 Gb3 localization in perfusion-Wxed murine central nervous system tissue. a and b Tissue was dehydrated with acetone before paraYnization. Tissue probed with anti-Gb3/CD77 antibodies (a) shows Gb3 concentrated within neurons; nonspeciWc reactions are absent from tissue treated with isotype control antibodies (b). c and d Ethanol dehydrated tissue probed with anti-Gb3/CD77 antibodies (c) show faint localization of Gb3 with neurons (arrows) in comparison to acetone treated tissue. Gb3 is not associated with vessels (asterisks) and nonspeciWc interactions are absent in tissue probed with isotype control antibodies (d). Scale bars indicate 100 m
Fig. 4 Gb3 localization in perfusion-Wxed, cryoprotected, free-Xoating sections of murine central nervous system tissue. In each frame, the white text box indicates the color that corresponds to a speciWc
antibody. Gb3 does not localize with CD31-positive endothelial cells (arrows) or GFAP-positive astrocytes. Scale bars indicate 10 m
kidney, whereas in the nervous system, it was associated with neurons. Notably, Gb3 did not colocalize with the endothelial cell marker CD31 in either of these tissue types. Immunohistochemical detection of lipids in paraYn sections presents a challenge because solvents used during tissue processing can alter the Wndings. The standard solvents used processing tissue for paraYn embedding are ethanol and xylene. Although the overall tissue morphology is well preserved during processing, ethanol extracts lipids, including Gb3, from the sample. Our data suggest that ethanol extraction of renal lipids may alter tissue antigenicity thereby eVecting anti-Gb3/CD77 and isotype control antibody recognition of epitopes. On the other hand, dehydration of tissue in acetone preserves a number of lipids, likely because of decreased lipid solubility in the solvent (Chark et al. 2004; Elleder and Lojda 1973). Interestingly, ethanol dehydration markedly reduced overall levels of renal Gb3 but only minimally decreased levels of neuronal Gb3. This result may be
explained partly by the distinct glycolipid proWles of nervous and renal tissue (Penick et al. 1966; Rauvala 1976; Watanabe and Nishiyama 1995); such diVerences in lipid composition may eVect the membrane environment and alter lipid solubility and extraction eYciency (Christie 1993). The lipid composition of membranes may also aVect tissue architecture during freezing. The free-Xoating staining method used perfusion-Wxed, cryoprotected, tissue from the kidney and nervous system. Similar staining patterns between acetone-dehydrated, paraYn-embedded sections and free-Xoating sections suggested that both methods reliably detected Gb3. Our results from murine renal tissue are in agreement with those of previous studies that examined murine renal Gb3 using Shiga toxin-binding assays to detect the glycolipid (Mattocks et al. 2006; Rutjes et al. 2002; Tesh et al. 1993). Similarly, our Wnding that neurons in the CNS express Gb3 is supported by a study that detected Gb3 on murine dorsal root ganglion neurons (Ren et al. 1999).
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Fig. 5 Gb3 localization in immersion-Wxed, murine kidney tissue. a–c Free-Xoating tissue sections depict Gb3 as green, CD31 as red, and DAPI as blue. a Gb3 localizes in tubules (asterisk) and glomerular structures. b Enlarged view of the boxed area from part (a) shows intravessel localization of Gb3 (arrow); c however, a similar pattern is apparent in tissue probed with isotype control antibodies (arrows) suggestive of a false-positive reaction. Scale bars indicate 10 m (a, c) or 5 m (b)
Fig. 6 Gb3 localization in immersion-Wxed, murine central nervous system tissue. In each frame, the white text box indicates the color that corresponds to a speciWc antibody. a Gb3 colocalizes with CD31(endothelial cell marker) (arrowheads). b Sections probed with isotype control antibody shows nonspeciWc recognition of vessel-like structures. c and d ParaYnembedded tissue sections show Gb3 localization with vessels (asterisks) and neurons (arrows) (c); however, vessels also were identiWed when using the isotype control antibody (d) indicating a false-positive reaction. Scale bars indicate 10 m (a, b) or 100 m (c, d)
Endothelial cells in the kidney and nervous system of normal C57BL/6 mice do not express Gb3. However, sections of immersion-Wxed tissue used for free-Xoating
staining had the appearance of Gb3 localization with the endothelium. The false-positive staining of the endothelium could be attributed partly to residual material (e.g., mouse
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immunoglobulins recognized by unadsorbed anti-rat IgM) in immersion-Wxed tissues. In conclusion, we have described a method of embedding tissue in paraYn that preserves tissue morphology and antigenicity of the neutral glycolipid, Gb3. This technique oVers results similar to those of free-Xoating, frozen sections. These methods may be useful for detection of other lipid antigens, although rigorous steps should be taken to verify that the dehydration solvents do not extract the lipid of interest and that the Wxation method does not cause falsepositive results. In depth studies examining diVerences in Gb3 expression in human and murine model systems are ongoing in our laboratory. Acknowledgments We thank Dr. C. A. Lingwood (Hospital for Sick Kids, Toronto, Ontario, Canada) and Dr. C. M. Thorpe (Tufts Medical Center, Boston, Massachusetts) for graciously providing the antiStx1B polyclonal antibody and Stx1B, respectively. Tissue preparation was performed by the University of Virginia Research Histology Core Facility. We also thank Dr. June Oshiro and Mitchell Psotka for critical review of the manuscript. This work was supported by funding from United States Public Health Service grant AI024431 to TGO. GLK was supported by a fellowship from the Biodefense Research Training and Career Development Grant T32 A1055432-01 from the National Institutes of Health
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