Immunohistochemical detection of sphingosine-1-phosphate receptor 1 and 5 in human multiple sclerosis lesions
Article first published online: 1 JUL 2014
© 2013 British Neuropathological Society
Neuropathology and Applied Neurobiology
Volume 40, Issue 5, pages 564–578, August 2014
How to Cite
Brana, C., Frossard, M. J., Pescini Gobert, R., Martinier, N., Boschert, U. and Seabrook, T. J. (2014), Immunohistochemical detection of sphingosine-1-phosphate receptor 1 and 5 in human multiple sclerosis lesions. Neuropathology and Applied Neurobiology, 40: 564–578. doi: 10.1111/nan.12048
- Issue published online: 1 JUL 2014
- Article first published online: 1 JUL 2014
- Accepted manuscript online: 31 MAR 2013 09:10PM EST
- Manuscript Accepted: 8 MAR 2013
- Manuscript Received: 26 SEP 2012
Figure S1. MS lesion characterization. MS lesions were characterized by the loss of MBP (A, B, Aia, Aiia, Bia) in both the white and grey matter and CD68+ macrophages/activated microglia (Aib, Aiib, Bib). In A, two white matter lesions (boxes marked i, ii shown at higher magnification) are seen in the MS7 case block. Lesion Ai shows a chronic active lesion with an irregular edge, myelin debris within macrophages (Aia) and CD68+ macrophages (Aib). On the right side of the tissue block there is a chronic inactive lesion (Aii) characterized by a more defined edge and lack of myelin debris (Aiia) and macrophages (Aiib). On the MS5 case block, a large Type 1 GM lesion is observed (B), with the GM/WM border delineated by the dashed line. The GM plaque is chronic active as there is myelin debris (Bia) and CD68+ macrophages (Bib) present in the lesion. The asterisk shows a chronic active lesion within the WM. Scale bars A and B = 1 mm, others 50 μm.
Figure S2. S1P1 and S1P5 Western blotting. To assess the specificity of the S1P antibodies on Western blot, we used CHO cells overexpressing S1P1 and S1P5 receptors. Cells were lysed and homogenized followed by centrifugation. Protein concentrations were measured using BCA protein assay (Thermo Scientific, Langenselbold, Germany). Equal amounts of protein (5 μg) were separated on 4–12% Tris-Bis electrophoresis gel (Invitrogen AG, Basel, Switzerland) and transferred to membranes (Invitrogen AG). After blocking membranes were incubated with either anti-S1P1 (1:500) or anti-S1P5 (1:500) overnight and primary antibodies detected by incubation with appropriate secondary antibodies. A band of the expected weigh molecular on Western blot further confirms the specificity of the S1P1 and S1P5 antibodies used for these studies.
Figure S3. S1P5 receptor expression in myelin. β-Galactosidase staining of tissue (A): expression of S1P5 was assessed using a S1P5+/LacZ mouse in which the entire genomic region was replaced by a cassette containing the coding region of the β-galactosidase (Lac-Z) gene. Cryosections (10 μm) were stained using a X-Gal staining kit (Genlantis, San Diego, CA, USA) according to the manufacturer's instructions. After colour development, the slides were washed in PBS and counterstained with Neutral Red before coverslipping. Data have showed abundant staining in myelinated tracts and less staining in the cortex and other grey matter areas. Triple immunofluorescence images demonstrate that S1P5 was not localized with blood vessel (Collagen IV, B) but was associated with myelin (MBP, C). Scales bars 100 μm (A), 20 μm (B), 10 μm (C).
Figure S4. S1P1 and S1P5 mRNA expression in MS lesions. RNA isolation and quantification by qPCR: frozen sections from tissue samples were collected in tubes. Total RNA was prepared with TRIzol Reagent (Invitrogen AG, Basel, Switzerland) using the manufacturer's protocol. One microgram of total RNA was used as template for reverse transcription with qScript cDNA Supermix (Quanta Biosciences, Inc., Gaithersburg, MD, USA). cDNA was diluted 20× and real-time PCR reaction was performed in triplicates using FastStart Universal SYBR Green Master mix (Roche, Rotkreuz, Switzerland) and the respective QuantiTect primer assays from QIAGEN (Hombrechtikon, Switzerland) on the LightCycler 480 Real-Time PCR System (Roche). Graph illustrates the relative quantification of human S1P1 and S1P5 mRNA in WM and GM lesions compared with NDC and WM controls. NDC, nondemented control; WM, white matter control; WM-MS, white matter in MS lesion; GM-MS, grey matter in MS lesion.
Table S1. Subject information for tissues used for immunohistochemistry.
Table S2. Multiple Sclerosis lesion classification and numbers.
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