Potassium (KV) Channels

Paraffin-embedded tissue immunoblot analysis was used to discriminate between prion disease-specific proteinase K-resistant PrPSc and cellular PrPC as described58

Paraffin-embedded tissue immunoblot analysis was used to discriminate between prion disease-specific proteinase K-resistant PrPSc and cellular PrPC as described58. For light microscopy, haematoxylin was used as a counterstain and sections imaged on a Ni.1 microscope (Nikon). tested the hypothesis that MZ B cells also play a role in the initial shuttling of prions from the blood-stream to FDC. MZ B cells were temporarily depleted from the MZ by antibody-mediated blocking of integrin function. We show that depletion of MZ Nuciferine B cells around the time of IV prion exposure did not affect the early accumulation of blood-borne prions upon splenic FDC or reduce susceptibility to IV prion infection. In conclusion, our data suggest that the initial delivery of blood-borne prions to FDC in the spleen occurs independently of MZ B cells. mouse experiments were obtained from The Roslin Institutes and University of Edinburghs ethics committees. All the experiments in this study were undertaken in accordance with the guidelines and regulations of the UK Home Office Animals (scientific procedures) Act 1986 and were performed under the authority of UK Home Office Project Licence PPL60/4325. Appropriate care was given to reduce harm and suffering, with anaesthesia was administered where necessary. At the end of the experiments the mice were humanely culled by cervical dislocation. Mice Female C57BL/6?J mice were obtained from Charles River Laboratories (Charles River, Margate, UK) and housed under specific pathogen-free conditions with a 12:12?h light:dark cycle. Food and water were provided anti-integrin antibody treatment Transient displacement of MZ B cells was achieved by IV injection with 100?g each of rat anti-mouse LFA-1 mAb (CD11a, clone M17/4, IgG2a) and rat anti-mouse integrin 4 mAb (CD49d, clone R1-2, IgG2b) as described previously28C30. Where indicated some mice were injected with non-specific rat IgG2a (clone eBR2a) and rat IgG2b (clone eB149/10H5) as isotype controls. All these antibodies were purchased from ThermoFisher (Loughborough, UK). Flow cytometry Single spleen cell suspensions were prepared and red blood cells lysed using red blood cell lysis buffer (Sigma, Poole, UK). Viable cells were counted and re-suspended in FACS buffer (PBS pH 7.4 containing 0.1% BSA, 0.1% sodium azide and 0.02% EDTA). Non-specific immunoglobulin-binding was blocked using Mouse Seroblock FcR (Bio-Rad Laboratories Watford, UK) and cells subsequently immunostained with the following mAb purchased from BioLegend (London, UK): anti-mouse CD1d-PerCP/Cy5.5 (clone Ly-38); anti-mouse CD21/35-Pacific Blue (clone 7G6); anti-mouse CD45R:B220-APC (clone RA3-6B2). Relevant non-specific antibody isotypes were used as controls. Cells were analysed on a LSR Fortessa with DIVA software (BD Biosciences). Cells were gated on lymphocytes, doublets excluded and data analysed with FlowJo software (FlowJo, LLC, Ashland OR, USA). Intravenous prion infection Mice were injected IV with 20?l of a 0.1% (weight/volume) brain homogenate prepared from mice terminally infected with TSLPR ME7 scrapie prions (containing approximately 1??103 ID50 units). The mice were then coded, Nuciferine and assessed blindly for the clinical signs of prion disease by independent husbandry technicians. Mice were culled at a standard clinical endpoint as described55. The clinical status of each mouse was confirmed by histopathological assessment of the prion disease-specific spongiform vacuolation in haematoxylin and eosin stained brain sections as described56. Immunohistochemistry Snap-frozen spleens were embedded in optimal cryotomy temperature compound and cryosectioned at 10 m thickness. Sections were then immunostained using the following antibodies: Nuciferine rat anti-mouse CD1d (clone 1B1; Bio-Rad Laboratories); rat anti-mouse CD21/35 (clone 7G6; BD Biosciences); anti-mouse CD45R:B220 (clone RA3-6B2); anti-mouse CD169 (MOMA-1; Bio-Rad Laboratories); Alexa Fluor 488-conjugated anti-mouse IgD (clone 11C26?c.2a; Biolegend); Alexa Fluor 594-conjugated goat anti-mouse IgM ( chain; ThermoFisher); anti-mouse MARCO (clone ED31; Bio-Rad Laboratories); Armenian hamster anti-mouse SIGNR1 (clone 22D1; eBioscience). Where appropriate, binding of primary antibodies was detected using.

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