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Strings are cell surface structures. (A) Unlike most PrP27-30, FL PrP Sc strings do not reside in dextran-positive endosomes. ScGT1 were incubated with a fixable Alexa 555–dextran (2 h; 250 µg/ml; red) to saturate the endosomal system, fixed, permeabilized, denatured with 3 M GdnSCN, and stained with either core D13 (a–c) or N-terminal mAb 8B4 (d–f). Within the cell (a–c), most D13 colocalized with dextran (c), consistent with endocytic PrP27-30 localization. The dorsal z = 0 plane (d) contains strings but no dextran. Internalized dextran was first seen in the contiguous z = 300 nm plane within the cell interior (e). The two images are superposed in f. Images sharpened by <t>2D</t> <t>nearest</t> <t>neighbor</t> deconvolution. (B) In chilled ScGT1, strings are found exclusively on the cell surface. (Step 1) Live ScGT1 were stained at 8°C with 8B4 followed by RRX secondary Fabs (red). (Step 2) Cells were fixed and permeabilized, blocked with unlabeled anti-mouse Fabs, and restained with 8B4 followed by a secondary IgG (green). No additional strings structures were observed in the green (c) versus the red (b) channels, indicating no intracellular strings.
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Strings are cell surface structures. (A) Unlike most PrP27-30, FL PrP Sc strings do not reside in dextran-positive endosomes. ScGT1 were incubated with a fixable Alexa 555–dextran (2 h; 250 µg/ml; red) to saturate the endosomal system, fixed, permeabilized, denatured with 3 M GdnSCN, and stained with either core D13 (a–c) or N-terminal mAb 8B4 (d–f). Within the cell (a–c), most D13 colocalized with dextran (c), consistent with endocytic PrP27-30 localization. The dorsal z = 0 plane (d) contains strings but no dextran. Internalized dextran was first seen in the contiguous z = 300 nm plane within the cell interior (e). The two images are superposed in f. Images sharpened by <t>2D</t> <t>nearest</t> <t>neighbor</t> deconvolution. (B) In chilled ScGT1, strings are found exclusively on the cell surface. (Step 1) Live ScGT1 were stained at 8°C with 8B4 followed by RRX secondary Fabs (red). (Step 2) Cells were fixed and permeabilized, blocked with unlabeled anti-mouse Fabs, and restained with 8B4 followed by a secondary IgG (green). No additional strings structures were observed in the green (c) versus the red (b) channels, indicating no intracellular strings.
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nearest neighbor deconvolution algorithm  (universal imaging inc)
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Strings are cell surface structures. (A) Unlike most PrP27-30, FL PrP Sc strings do not reside in dextran-positive endosomes. ScGT1 were incubated with a fixable Alexa 555–dextran (2 h; 250 µg/ml; red) to saturate the endosomal system, fixed, permeabilized, denatured with 3 M GdnSCN, and stained with either core D13 (a–c) or N-terminal mAb 8B4 (d–f). Within the cell (a–c), most D13 colocalized with dextran (c), consistent with endocytic PrP27-30 localization. The dorsal z = 0 plane (d) contains strings but no dextran. Internalized dextran was first seen in the contiguous z = 300 nm plane within the cell interior (e). The two images are superposed in f. Images sharpened by <t>2D</t> <t>nearest</t> <t>neighbor</t> deconvolution. (B) In chilled ScGT1, strings are found exclusively on the cell surface. (Step 1) Live ScGT1 were stained at 8°C with 8B4 followed by RRX secondary Fabs (red). (Step 2) Cells were fixed and permeabilized, blocked with unlabeled anti-mouse Fabs, and restained with 8B4 followed by a secondary IgG (green). No additional strings structures were observed in the green (c) versus the red (b) channels, indicating no intracellular strings.
Nearest Neighbor Deconvolution Algorithm, supplied by universal imaging inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/nearest+neighbor+deconvolution/nearest+neighbor+deconvolution+algorithm/10__1074_slash_jbc__m202626200-136-3-13
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Image Search Results


Strings are cell surface structures. (A) Unlike most PrP27-30, FL PrP Sc strings do not reside in dextran-positive endosomes. ScGT1 were incubated with a fixable Alexa 555–dextran (2 h; 250 µg/ml; red) to saturate the endosomal system, fixed, permeabilized, denatured with 3 M GdnSCN, and stained with either core D13 (a–c) or N-terminal mAb 8B4 (d–f). Within the cell (a–c), most D13 colocalized with dextran (c), consistent with endocytic PrP27-30 localization. The dorsal z = 0 plane (d) contains strings but no dextran. Internalized dextran was first seen in the contiguous z = 300 nm plane within the cell interior (e). The two images are superposed in f. Images sharpened by 2D nearest neighbor deconvolution. (B) In chilled ScGT1, strings are found exclusively on the cell surface. (Step 1) Live ScGT1 were stained at 8°C with 8B4 followed by RRX secondary Fabs (red). (Step 2) Cells were fixed and permeabilized, blocked with unlabeled anti-mouse Fabs, and restained with 8B4 followed by a secondary IgG (green). No additional strings structures were observed in the green (c) versus the red (b) channels, indicating no intracellular strings.

Journal: The Journal of Cell Biology

Article Title: Live imaging of prions reveals nascent PrP Sc in cell-surface, raft-associated amyloid strings and webs

doi: 10.1083/jcb.201308028

Figure Lengend Snippet: Strings are cell surface structures. (A) Unlike most PrP27-30, FL PrP Sc strings do not reside in dextran-positive endosomes. ScGT1 were incubated with a fixable Alexa 555–dextran (2 h; 250 µg/ml; red) to saturate the endosomal system, fixed, permeabilized, denatured with 3 M GdnSCN, and stained with either core D13 (a–c) or N-terminal mAb 8B4 (d–f). Within the cell (a–c), most D13 colocalized with dextran (c), consistent with endocytic PrP27-30 localization. The dorsal z = 0 plane (d) contains strings but no dextran. Internalized dextran was first seen in the contiguous z = 300 nm plane within the cell interior (e). The two images are superposed in f. Images sharpened by 2D nearest neighbor deconvolution. (B) In chilled ScGT1, strings are found exclusively on the cell surface. (Step 1) Live ScGT1 were stained at 8°C with 8B4 followed by RRX secondary Fabs (red). (Step 2) Cells were fixed and permeabilized, blocked with unlabeled anti-mouse Fabs, and restained with 8B4 followed by a secondary IgG (green). No additional strings structures were observed in the green (c) versus the red (b) channels, indicating no intracellular strings.

Article Snippet: In some cases (indicated in the relevant figures), nearest neighbor 2D deconvolution (MetaMorph) was used to enhance the signal/noise ratio.

Techniques: Incubation, Staining