bioimagesUnknownAlexander RouvinskiThe Journal of Cell Biologyhttps://www.ebi.ac.uk/biostudies/studies/S-JCBD-201308028JCB

ScGT1/RML cells were fixed with formaldehyde, denatured with GdnSCN, and then stained with mAb SAF32 followed by an RRX-labeled secondary antibody. Cells were photographed using an Axiovert 200M microscope using a 40x NA1.3 Plan Neofluar oil objective and a Zeiss FS43HE filter cube. Images were acquired using the MetaMorph 6.2 software. Twenty six micrographs at 300nm z increments were included in this z-stack. A fragment of plane # 9 of 26 is shown in Fig. S1B in the paper. Notice the presence of strings in most cells in the field. Of note, strings are best observed using a 100x objective, but we chose a low magnification here to image a large number of cells.

(This image is associated with, but not shown in, the paper's Fig. 2C,b.) ScGT1 cells growth-arrested by a 6d MMC/BrdU treatment were processed using the FA-denaturing protocol and co-stained with mAb SAF32 and with mAb 8H4 followed by subtype-specific secondary antibodies: cy2 anti-IgG2b for SAF32 and RRX anti-IgG1 for 8H4 (white). Results for 8H4 are shown in this stack (white). Cells were photographed using an Axiovert 200M microscope using a 100x NA1.3 Plan Neofluar oil objective and a Zeiss FS43HE filter cube. Images were acquired using the MetaMorph 6.2 software. Thirty six micrographs at 300nm z increments were included in this z-stack. In addition to webs of branching PrP strings covering large areas of the cell periphery, this core Ab also stains extensive intracellular dots that

ScGT1 cells growth-arrested by a 6d MMC/BrdU treatment were processed using the FA-denaturing protocol and co-stained with mAb SAF32 and with mAb 8H4 followed by subtype-specific secondary antibodies: cy2 anti-IgG2b for SAF32 (white) and RRX anti-IgG1 for 8H4. Results for SAF32 are shown in this stack. Cells were photographed using an Axiovert 200M microscope using a 100x NA1.3 Plan Neofluar oil objective and a Zeiss FS38HE filter cube. Images were acquired using the MetaMorph 6.2 software. Thirty five micrographs at 300nm z increments were included in this z-stack. This stack shows that webs of branching PrP strings cover large areas of the cell periphery; elsewhere, the paper further shows that strings are found on the cell surface. Plane #22 of 35 is shown in Fig. 1D,a. Please notice th

Chandler-infected SMB cells were fixed with formaldehyde, permeabilized with Triton X-100, and then stained with mAb 8B4 followed by an RRX-labeled secondary Ab (white). Cells were photographed using an Axiovert 200M microscope using a 100x NA1.3 Plan Neofluar oil objective and a Zeiss FS43HE filter cube. Images were acquired using the MetaMorph 6.2 software. Sixteen micrographs at 300nm z increments were included in this z-stack. PrP strings are clearly seen in several cells in the field at various z depths but are most prominent in the cell in the center of the field in planes 7-11. A fragment of plane #9 of 16 is shown in Fig. 1C,e. Please notice that a 3-D deconvolved image of this stack also appears in the paper in Fig. 6B.

ScGT1 cells growth-arrested by a 6d MMC/BrdU treatment were processed using the FA-denaturing protocol and co-stained with mAb SAF32 and with mAb 8H4 followed by subtype-specific secondary antibodies: cy2 anti-IgG2b for SAF32 (white) and RRX anti-IgG1 for 8H4. Results for SAF32 are shown in this stack. Cells were photographed using an Axiovert 200M microscope using a 100x NA1.3 Plan Neofluar oil objective and a Zeiss FS38HE filter cube. Thirty five images at 300nm z increments were acquired using the MetaMorph 6.2 software. The z-stack was processed by blind 3D deconvolution using the Huygens software (Scientific Volume Imaging). Twenty sequential planes from the 3D deconvolved stack are included in the data presented here. Webs of branching PrP strings cover large areas of the cell periph

ScGT1 cells growth-arrested by a 6d MMC/BrdU treatment were processed using the FA denaturing protocol and co-stained with mAb SAF32 and with mAb 8H4 followed by subtype-specific secondary antibodies: cy2 anti-IgG2b for SAF32 and RRX anti-IgG1 for 8H4. Results for 8H4 are shown in this stack (white). Cells were photographed using an Axiovert 200M microscope using a 100x NA1.3 Plan Neofluoar oil objective and a Zeiss FS43HE filter cube. Thirty six images at 300nm z increments were acquired using the MetaMorph 6.2 software. The z-stack was processed by blind 3D deconvolution using the Huygens software (Scientific Volume Imaging). Twenty sequential planes from the 3D deconvolved stack are included in the data presented here. In addition to webs of PrP strings covering large areas of the cell

SMB-PPS cells were fixed with formaldehyde, permeabilized with Triton X-100, and then stained with mAb 8B4 followed by an RRX-labeled secondary Ab (white). Cells were photographed using an Axiovert 200M microscope using a 100x NA1.3 Plan Neofluar oil objective and a Zeiss FS43HE filter cube. Images were acquired using the MetaMorph 6.2 software. Thirty one micrographs at 300nm z increments were included in this z-stack. The diffuse cellular signal represents PrPC. A fragment of plane #9 of 31 is shown in Fig. 1C,d.

ScGT1 cells growth-arrested by a 6d MMC/BrdU treatment were processed using the FA-denaturing protocol and co-stained with mAb SAF32 and with mAb 8H4 followed by subtype-specific secondary antibodies: cy2 anti-IgG2b for SAF32 (red) and RRX anti-IgG1 for 8H4 (green). Superposed results with both antibodies are shown in this stack. Cells were photographed using an Axiovert 200M microscope using a 100x NA1.3 oil objective and Zeiss FS38HE and FS43HE filter cubes. Images at 300nm z increments were acquired using the MetaMorph 6.2 software. The two z-stacks (one for each channel) were independently processed by blind 3D deconvolution using the Huygens software (Scientific Volume Imaging). The ten most relevant sequential planes were then superposed using MetaMorph. Both antibodies co-decorate w

bioimagesImage 137278 (Figure 2 - C,b - raw data)Image 137267 (Figure 1 - C,d)Figure 2 - C,bImage 137281 (Figure 2 - C,b)Figure 2 - C,cImage 137292 (Figure 2 - C,c)Supplemental Figure 1 - BFigure 2 - C,b - raw dataSupplemental Figure 1Image 137268 (Figure 1 - C,e)Image 137282 (Figure 1 - D,b)Figure 1 - D,aFigure 1 - C,dFigure 1 - D,bFigure 1 - C,eImage 137289 (Supplemental Figure 1 - B)Image 137276 (Figure 1 - D,a)Figure 1Figure 2Sanaa MoussaGeorge A. CarlsonSusan F. GodsaveCarsten KorthMaria KouninJanine KutzscheDulce Papy-GarciaKatarina LuhrMiri D. GoldbergPeter J. PetersSharon KarnielyKrister KristenssonAlbert TaraboulosGabriela WarburgRoman LyakhovetskyAlexander RouvinskifalseLive imaging of prions reveals nascent PrPScin cell-surface, raft-associated amyloid strings and webs2014-02-03T11:25:54Z2018-11-29T11:25:54Z2018-11-29T11:25:54ZS-JCBD-20130802810.1083/jcb.201308028