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HCR-Proxy design and implementation. ( A ) Schematic overview of HCR-Proxy. The main part of the in situ workflow is streamlined into tubes, where chemically fixed cells are hybridized with pairs of antisense probes towards target RNA. Only the complete probe pair alignment enables signal amplification (HCR) via polymerized DIG-labelled metastable hairpins and therefore recruitment of PL enzyme to catalyse in situ proximity biotinylation (Proxy). ( B ) HCR-FISH IF micrographs of non-coding RNA Malat1 (yellow, DyLight594-conjugated anti-DIG) in mESCs co-localizing with the nuclear speckle <t>marker</t> <t>SC-35</t> (red, SC-35 antibody), merged with DAPI staining (blue). The co-localization of the signal was calculated using Pearson’s correlation coefficient (PCC) from 13 images (dots represent each cell). ( C ) HCR-Proxy IF micrographs of Malat1 in mESCs supported with signal intensity profiles of RNA FISH (red, DyLight594-conjugated anti-DIG) and HCR-Proxy (yellow, Alexa647-conjugated streptavidin) signals. Cell nuclei were visualized with DAPI staining (blue). The co-localization of the signal was calculated using PCC from 10 images (dots represent each cell). ( D ) HCR-Proxy IF micrographs of the Efl1 intronic transcript (red, DyLight594-conjugated anti-DIG) and its proximal vicinity labelled with two different PL enzymes, APEX2 or TurboID (yellow, Alexa647-conjugated streptavidin), merged with DAPI staining (blue). The difference in intensity density was calculated with two-sided Student’s t -test ( ***P < 0.001, **P < 0.01, *P < 0.05). ( E ) HCR-Proxy IF micrographs of the Efl1 intronic transcript with signal intensity profiles of RNA FISH (red, DyLight594-conjugated anti-DIG) and HCR-Proxy (yellow, Alexa Fluor 647-conjugated streptavidin) signals, merged with DAPI staining (blue).
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HCR-Proxy design and implementation. ( A ) Schematic overview of HCR-Proxy. The main part of the in situ workflow is streamlined into tubes, where chemically fixed cells are hybridized with pairs of antisense probes towards target RNA. Only the complete probe pair alignment enables signal amplification (HCR) via polymerized DIG-labelled metastable hairpins and therefore recruitment of PL enzyme to catalyse in situ proximity biotinylation (Proxy). ( B ) HCR-FISH IF micrographs of non-coding RNA Malat1 (yellow, DyLight594-conjugated anti-DIG) in mESCs co-localizing with the nuclear speckle <t>marker</t> <t>SC-35</t> (red, SC-35 antibody), merged with DAPI staining (blue). The co-localization of the signal was calculated using Pearson’s correlation coefficient (PCC) from 13 images (dots represent each cell). ( C ) HCR-Proxy IF micrographs of Malat1 in mESCs supported with signal intensity profiles of RNA FISH (red, DyLight594-conjugated anti-DIG) and HCR-Proxy (yellow, Alexa647-conjugated streptavidin) signals. Cell nuclei were visualized with DAPI staining (blue). The co-localization of the signal was calculated using PCC from 10 images (dots represent each cell). ( D ) HCR-Proxy IF micrographs of the Efl1 intronic transcript (red, DyLight594-conjugated anti-DIG) and its proximal vicinity labelled with two different PL enzymes, APEX2 or TurboID (yellow, Alexa647-conjugated streptavidin), merged with DAPI staining (blue). The difference in intensity density was calculated with two-sided Student’s t -test ( ***P < 0.001, **P < 0.01, *P < 0.05). ( E ) HCR-Proxy IF micrographs of the Efl1 intronic transcript with signal intensity profiles of RNA FISH (red, DyLight594-conjugated anti-DIG) and HCR-Proxy (yellow, Alexa Fluor 647-conjugated streptavidin) signals, merged with DAPI staining (blue).
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HCR-Proxy design and implementation. ( A ) Schematic overview of HCR-Proxy. The main part of the in situ workflow is streamlined into tubes, where chemically fixed cells are hybridized with pairs of antisense probes towards target RNA. Only the complete probe pair alignment enables signal amplification (HCR) via polymerized DIG-labelled metastable hairpins and therefore recruitment of PL enzyme to catalyse in situ proximity biotinylation (Proxy). ( B ) HCR-FISH IF micrographs of non-coding RNA Malat1 (yellow, DyLight594-conjugated anti-DIG) in mESCs co-localizing with the nuclear speckle <t>marker</t> <t>SC-35</t> (red, SC-35 antibody), merged with DAPI staining (blue). The co-localization of the signal was calculated using Pearson’s correlation coefficient (PCC) from 13 images (dots represent each cell). ( C ) HCR-Proxy IF micrographs of Malat1 in mESCs supported with signal intensity profiles of RNA FISH (red, DyLight594-conjugated anti-DIG) and HCR-Proxy (yellow, Alexa647-conjugated streptavidin) signals. Cell nuclei were visualized with DAPI staining (blue). The co-localization of the signal was calculated using PCC from 10 images (dots represent each cell). ( D ) HCR-Proxy IF micrographs of the Efl1 intronic transcript (red, DyLight594-conjugated anti-DIG) and its proximal vicinity labelled with two different PL enzymes, APEX2 or TurboID (yellow, Alexa647-conjugated streptavidin), merged with DAPI staining (blue). The difference in intensity density was calculated with two-sided Student’s t -test ( ***P < 0.001, **P < 0.01, *P < 0.05). ( E ) HCR-Proxy IF micrographs of the Efl1 intronic transcript with signal intensity profiles of RNA FISH (red, DyLight594-conjugated anti-DIG) and HCR-Proxy (yellow, Alexa Fluor 647-conjugated streptavidin) signals, merged with DAPI staining (blue).
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HCR-Proxy design and implementation. ( A ) Schematic overview of HCR-Proxy. The main part of the in situ workflow is streamlined into tubes, where chemically fixed cells are hybridized with pairs of antisense probes towards target RNA. Only the complete probe pair alignment enables signal amplification (HCR) via polymerized DIG-labelled metastable hairpins and therefore recruitment of PL enzyme to catalyse in situ proximity biotinylation (Proxy). ( B ) HCR-FISH IF micrographs of non-coding RNA Malat1 (yellow, DyLight594-conjugated anti-DIG) in mESCs co-localizing with the nuclear speckle <t>marker</t> <t>SC-35</t> (red, SC-35 antibody), merged with DAPI staining (blue). The co-localization of the signal was calculated using Pearson’s correlation coefficient (PCC) from 13 images (dots represent each cell). ( C ) HCR-Proxy IF micrographs of Malat1 in mESCs supported with signal intensity profiles of RNA FISH (red, DyLight594-conjugated anti-DIG) and HCR-Proxy (yellow, Alexa647-conjugated streptavidin) signals. Cell nuclei were visualized with DAPI staining (blue). The co-localization of the signal was calculated using PCC from 10 images (dots represent each cell). ( D ) HCR-Proxy IF micrographs of the Efl1 intronic transcript (red, DyLight594-conjugated anti-DIG) and its proximal vicinity labelled with two different PL enzymes, APEX2 or TurboID (yellow, Alexa647-conjugated streptavidin), merged with DAPI staining (blue). The difference in intensity density was calculated with two-sided Student’s t -test ( ***P < 0.001, **P < 0.01, *P < 0.05). ( E ) HCR-Proxy IF micrographs of the Efl1 intronic transcript with signal intensity profiles of RNA FISH (red, DyLight594-conjugated anti-DIG) and HCR-Proxy (yellow, Alexa Fluor 647-conjugated streptavidin) signals, merged with DAPI staining (blue).
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HCR-Proxy design and implementation. ( A ) Schematic overview of HCR-Proxy. The main part of the in situ workflow is streamlined into tubes, where chemically fixed cells are hybridized with pairs of antisense probes towards target RNA. Only the complete probe pair alignment enables signal amplification (HCR) via polymerized DIG-labelled metastable hairpins and therefore recruitment of PL enzyme to catalyse in situ proximity biotinylation (Proxy). ( B ) HCR-FISH IF micrographs of non-coding RNA Malat1 (yellow, DyLight594-conjugated anti-DIG) in mESCs co-localizing with the nuclear speckle <t>marker</t> <t>SC-35</t> (red, SC-35 antibody), merged with DAPI staining (blue). The co-localization of the signal was calculated using Pearson’s correlation coefficient (PCC) from 13 images (dots represent each cell). ( C ) HCR-Proxy IF micrographs of Malat1 in mESCs supported with signal intensity profiles of RNA FISH (red, DyLight594-conjugated anti-DIG) and HCR-Proxy (yellow, Alexa647-conjugated streptavidin) signals. Cell nuclei were visualized with DAPI staining (blue). The co-localization of the signal was calculated using PCC from 10 images (dots represent each cell). ( D ) HCR-Proxy IF micrographs of the Efl1 intronic transcript (red, DyLight594-conjugated anti-DIG) and its proximal vicinity labelled with two different PL enzymes, APEX2 or TurboID (yellow, Alexa647-conjugated streptavidin), merged with DAPI staining (blue). The difference in intensity density was calculated with two-sided Student’s t -test ( ***P < 0.001, **P < 0.01, *P < 0.05). ( E ) HCR-Proxy IF micrographs of the Efl1 intronic transcript with signal intensity profiles of RNA FISH (red, DyLight594-conjugated anti-DIG) and HCR-Proxy (yellow, Alexa Fluor 647-conjugated streptavidin) signals, merged with DAPI staining (blue).
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HCR-Proxy design and implementation. ( A ) Schematic overview of HCR-Proxy. The main part of the in situ workflow is streamlined into tubes, where chemically fixed cells are hybridized with pairs of antisense probes towards target RNA. Only the complete probe pair alignment enables signal amplification (HCR) via polymerized DIG-labelled metastable hairpins and therefore recruitment of PL enzyme to catalyse in situ proximity biotinylation (Proxy). ( B ) HCR-FISH IF micrographs of non-coding RNA Malat1 (yellow, DyLight594-conjugated anti-DIG) in mESCs co-localizing with the nuclear speckle <t>marker</t> <t>SC-35</t> (red, SC-35 antibody), merged with DAPI staining (blue). The co-localization of the signal was calculated using Pearson’s correlation coefficient (PCC) from 13 images (dots represent each cell). ( C ) HCR-Proxy IF micrographs of Malat1 in mESCs supported with signal intensity profiles of RNA FISH (red, DyLight594-conjugated anti-DIG) and HCR-Proxy (yellow, Alexa647-conjugated streptavidin) signals. Cell nuclei were visualized with DAPI staining (blue). The co-localization of the signal was calculated using PCC from 10 images (dots represent each cell). ( D ) HCR-Proxy IF micrographs of the Efl1 intronic transcript (red, DyLight594-conjugated anti-DIG) and its proximal vicinity labelled with two different PL enzymes, APEX2 or TurboID (yellow, Alexa647-conjugated streptavidin), merged with DAPI staining (blue). The difference in intensity density was calculated with two-sided Student’s t -test ( ***P < 0.001, **P < 0.01, *P < 0.05). ( E ) HCR-Proxy IF micrographs of the Efl1 intronic transcript with signal intensity profiles of RNA FISH (red, DyLight594-conjugated anti-DIG) and HCR-Proxy (yellow, Alexa Fluor 647-conjugated streptavidin) signals, merged with DAPI staining (blue).
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Image Search Results


HCR-Proxy design and implementation. ( A ) Schematic overview of HCR-Proxy. The main part of the in situ workflow is streamlined into tubes, where chemically fixed cells are hybridized with pairs of antisense probes towards target RNA. Only the complete probe pair alignment enables signal amplification (HCR) via polymerized DIG-labelled metastable hairpins and therefore recruitment of PL enzyme to catalyse in situ proximity biotinylation (Proxy). ( B ) HCR-FISH IF micrographs of non-coding RNA Malat1 (yellow, DyLight594-conjugated anti-DIG) in mESCs co-localizing with the nuclear speckle marker SC-35 (red, SC-35 antibody), merged with DAPI staining (blue). The co-localization of the signal was calculated using Pearson’s correlation coefficient (PCC) from 13 images (dots represent each cell). ( C ) HCR-Proxy IF micrographs of Malat1 in mESCs supported with signal intensity profiles of RNA FISH (red, DyLight594-conjugated anti-DIG) and HCR-Proxy (yellow, Alexa647-conjugated streptavidin) signals. Cell nuclei were visualized with DAPI staining (blue). The co-localization of the signal was calculated using PCC from 10 images (dots represent each cell). ( D ) HCR-Proxy IF micrographs of the Efl1 intronic transcript (red, DyLight594-conjugated anti-DIG) and its proximal vicinity labelled with two different PL enzymes, APEX2 or TurboID (yellow, Alexa647-conjugated streptavidin), merged with DAPI staining (blue). The difference in intensity density was calculated with two-sided Student’s t -test ( ***P < 0.001, **P < 0.01, *P < 0.05). ( E ) HCR-Proxy IF micrographs of the Efl1 intronic transcript with signal intensity profiles of RNA FISH (red, DyLight594-conjugated anti-DIG) and HCR-Proxy (yellow, Alexa Fluor 647-conjugated streptavidin) signals, merged with DAPI staining (blue).

Journal: Nucleic Acids Research

Article Title: HCR-Proxy resolves site-specific proximal RNA microenvironments at subcompartmental resolution

doi: 10.1093/nar/gkag086

Figure Lengend Snippet: HCR-Proxy design and implementation. ( A ) Schematic overview of HCR-Proxy. The main part of the in situ workflow is streamlined into tubes, where chemically fixed cells are hybridized with pairs of antisense probes towards target RNA. Only the complete probe pair alignment enables signal amplification (HCR) via polymerized DIG-labelled metastable hairpins and therefore recruitment of PL enzyme to catalyse in situ proximity biotinylation (Proxy). ( B ) HCR-FISH IF micrographs of non-coding RNA Malat1 (yellow, DyLight594-conjugated anti-DIG) in mESCs co-localizing with the nuclear speckle marker SC-35 (red, SC-35 antibody), merged with DAPI staining (blue). The co-localization of the signal was calculated using Pearson’s correlation coefficient (PCC) from 13 images (dots represent each cell). ( C ) HCR-Proxy IF micrographs of Malat1 in mESCs supported with signal intensity profiles of RNA FISH (red, DyLight594-conjugated anti-DIG) and HCR-Proxy (yellow, Alexa647-conjugated streptavidin) signals. Cell nuclei were visualized with DAPI staining (blue). The co-localization of the signal was calculated using PCC from 10 images (dots represent each cell). ( D ) HCR-Proxy IF micrographs of the Efl1 intronic transcript (red, DyLight594-conjugated anti-DIG) and its proximal vicinity labelled with two different PL enzymes, APEX2 or TurboID (yellow, Alexa647-conjugated streptavidin), merged with DAPI staining (blue). The difference in intensity density was calculated with two-sided Student’s t -test ( ***P < 0.001, **P < 0.01, *P < 0.05). ( E ) HCR-Proxy IF micrographs of the Efl1 intronic transcript with signal intensity profiles of RNA FISH (red, DyLight594-conjugated anti-DIG) and HCR-Proxy (yellow, Alexa Fluor 647-conjugated streptavidin) signals, merged with DAPI staining (blue).

Article Snippet: To validate co-localization with nuclear condensates and to confirm localization of candidate proteins, we used the following antibodies against: SC-35 (mouse, Santa Cruz Biotechnology, cat #sc-53518, 1:200 dilution), NPM1 (mouse, ThermoFisher Scientific, cat #32-5200, 1:100 dilution), FBL (rabbit, Abcam, cat #ab5821, 1:200 dilution), TXNRD1 (rabbit, Proteintech, cat #11117-1-AP, 1:200 dilution), and GFP (rabbit, ThermoFisher Scientific, cat #A-21311, 1:100 dilution).

Techniques: In Situ, Amplification, Marker, Staining