proteinase k New England Biolabs Search Results


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  • 96
    New England Biolabs thermolabile proteinase k
    Thermolabile Proteinase K, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher protease k
    IL-8 secretion induced by conditioned supernatants generated from Campylobacter -T84 cell coculture. (A) Polarized T84 cells were incubated for 24 h either with bacterium-free conditioned supernatants generated from the apical or basolateral supernatant of polarized T84 cells that had been inoculated with C. jejuni 81-176 for 4 h or with C. jejuni 81-176 alone cultured in the invasion medium for 4 h (bacterial). T84 cells were incubated with the bacterial culture medium in the apical chamber, with the apical conditioned supernatant in the apical chamber, or with the basolateral conditioned supernatant in the basolateral chamber. Polarized T84 cells inoculated apically with live C. jejuni 81-176 for 4 h and 24 h were used as controls. (B) The basolateral conditioned supernatant generated from C. jejuni 81-176-T84 cell coculture either was not pretreated (−) or was pretreated with either DNase I (10 U/ml) at 37°C for 2 h, polymyxin B (20 μg/ml) at 37°C for 30 min (PLXB), <t>protease</t> K (100 μg/ml) overnight followed by a 20-min incubation at 100°C (ProtK), or a 20-min incubation at 100°C without protease K (Boiling). Polarized T84 cells were incubated with the pretreated or untreated conditioned supernatants from wt C. jejuni 81-176-T84 cell coculture in the basolateral chamber at 37°C for 24 h. (C) Polarized T84 cells were treated basolaterally with a C. jejuni 81-176 DNA extract (25 μg/ml) (DNA) and DNase I-treated C. jejuni 81-176 DNA (DNA + DNase) at 37°C for 24 h. (D) Polarized T84 cells were incubated basolaterally with different concentrations of E. coli LPS in the presence or absence of 20 μg/ml PLXB. (E) Polarized T84 cells were incubated with conditioned supernatants from a coculture of T84 cells with wt C. jejuni 81-176 or its flaA , pflA , cdtB , or flaA cdtB mutant in the basolateral chamber at 37°C for 24 h. After different treatments, the apical (filled bars) and basolateral (open bars) media were collected. IL-8 concentrations were determined by ELISA. The data are means for three independent experiments; error bars, standard deviations. The data in panels B and E are expressed as the ratio of the IL-8 level induced by a treated basolateral conditioned supernatant to that induced by an untreated supernatant and as the ratio of the IL-8 level induced by a mutant basolateral conditioned supernatant to that induced by a wt supernatant. *, P
    Protease K, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 447 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs proteinase k new england biolabs cat
    IL-8 secretion induced by conditioned supernatants generated from Campylobacter -T84 cell coculture. (A) Polarized T84 cells were incubated for 24 h either with bacterium-free conditioned supernatants generated from the apical or basolateral supernatant of polarized T84 cells that had been inoculated with C. jejuni 81-176 for 4 h or with C. jejuni 81-176 alone cultured in the invasion medium for 4 h (bacterial). T84 cells were incubated with the bacterial culture medium in the apical chamber, with the apical conditioned supernatant in the apical chamber, or with the basolateral conditioned supernatant in the basolateral chamber. Polarized T84 cells inoculated apically with live C. jejuni 81-176 for 4 h and 24 h were used as controls. (B) The basolateral conditioned supernatant generated from C. jejuni 81-176-T84 cell coculture either was not pretreated (−) or was pretreated with either DNase I (10 U/ml) at 37°C for 2 h, polymyxin B (20 μg/ml) at 37°C for 30 min (PLXB), <t>protease</t> K (100 μg/ml) overnight followed by a 20-min incubation at 100°C (ProtK), or a 20-min incubation at 100°C without protease K (Boiling). Polarized T84 cells were incubated with the pretreated or untreated conditioned supernatants from wt C. jejuni 81-176-T84 cell coculture in the basolateral chamber at 37°C for 24 h. (C) Polarized T84 cells were treated basolaterally with a C. jejuni 81-176 DNA extract (25 μg/ml) (DNA) and DNase I-treated C. jejuni 81-176 DNA (DNA + DNase) at 37°C for 24 h. (D) Polarized T84 cells were incubated basolaterally with different concentrations of E. coli LPS in the presence or absence of 20 μg/ml PLXB. (E) Polarized T84 cells were incubated with conditioned supernatants from a coculture of T84 cells with wt C. jejuni 81-176 or its flaA , pflA , cdtB , or flaA cdtB mutant in the basolateral chamber at 37°C for 24 h. After different treatments, the apical (filled bars) and basolateral (open bars) media were collected. IL-8 concentrations were determined by ELISA. The data are means for three independent experiments; error bars, standard deviations. The data in panels B and E are expressed as the ratio of the IL-8 level induced by a treated basolateral conditioned supernatant to that induced by an untreated supernatant and as the ratio of the IL-8 level induced by a mutant basolateral conditioned supernatant to that induced by a wt supernatant. *, P
    Proteinase K New England Biolabs Cat, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs proteinase k proteinase k
    Forms of MIR2911 in cabbage extract. ( a ) Edible Plant Derived Exosome-like Nanoparticles (EPDENs) were purified from clarified cabbage lysate via ultracentrifugation. Levels of MIR2911 were quantified by qRT-PCR in various fractions: cabbage leaves liquefied in a blender (Liquefied Cabbage); Supernatant from 10,000 × g centrifugation (Slow Clarification); Supernatant from 150,000 × g ultracentrifugation (Ultra Clarification); EPDEN bands at 30% 45% interface (EPDEN band1) and 45% 60% interface (EPDEN band2) of sucrose gradient fractionation of total vesicles. ( b ) Size exclusion chromatography analysis of MIR2911 in ultracentrifugation-clarified cabbage lysate ( c ) qRT-PCR analysis of MIR2911 levels in proteinase K-treated or control (PBS-treated) ultracentrifugation-clarified cabbage lysate.
    Proteinase K Proteinase K, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 266 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene proteinase k
    Forms of MIR2911 in cabbage extract. ( a ) Edible Plant Derived Exosome-like Nanoparticles (EPDENs) were purified from clarified cabbage lysate via ultracentrifugation. Levels of MIR2911 were quantified by qRT-PCR in various fractions: cabbage leaves liquefied in a blender (Liquefied Cabbage); Supernatant from 10,000 × g centrifugation (Slow Clarification); Supernatant from 150,000 × g ultracentrifugation (Ultra Clarification); EPDEN bands at 30% 45% interface (EPDEN band1) and 45% 60% interface (EPDEN band2) of sucrose gradient fractionation of total vesicles. ( b ) Size exclusion chromatography analysis of MIR2911 in ultracentrifugation-clarified cabbage lysate ( c ) qRT-PCR analysis of MIR2911 levels in proteinase K-treated or control (PBS-treated) ultracentrifugation-clarified cabbage lysate.
    Proteinase K, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 193 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche proteinase k
    Forms of MIR2911 in cabbage extract. ( a ) Edible Plant Derived Exosome-like Nanoparticles (EPDENs) were purified from clarified cabbage lysate via ultracentrifugation. Levels of MIR2911 were quantified by qRT-PCR in various fractions: cabbage leaves liquefied in a blender (Liquefied Cabbage); Supernatant from 10,000 × g centrifugation (Slow Clarification); Supernatant from 150,000 × g ultracentrifugation (Ultra Clarification); EPDEN bands at 30% 45% interface (EPDEN band1) and 45% 60% interface (EPDEN band2) of sucrose gradient fractionation of total vesicles. ( b ) Size exclusion chromatography analysis of MIR2911 in ultracentrifugation-clarified cabbage lysate ( c ) qRT-PCR analysis of MIR2911 levels in proteinase K-treated or control (PBS-treated) ultracentrifugation-clarified cabbage lysate.
    Proteinase K, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 20739 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boehringer Mannheim proteinase k
    Forms of MIR2911 in cabbage extract. ( a ) Edible Plant Derived Exosome-like Nanoparticles (EPDENs) were purified from clarified cabbage lysate via ultracentrifugation. Levels of MIR2911 were quantified by qRT-PCR in various fractions: cabbage leaves liquefied in a blender (Liquefied Cabbage); Supernatant from 10,000 × g centrifugation (Slow Clarification); Supernatant from 150,000 × g ultracentrifugation (Ultra Clarification); EPDEN bands at 30% 45% interface (EPDEN band1) and 45% 60% interface (EPDEN band2) of sucrose gradient fractionation of total vesicles. ( b ) Size exclusion chromatography analysis of MIR2911 in ultracentrifugation-clarified cabbage lysate ( c ) qRT-PCR analysis of MIR2911 levels in proteinase K-treated or control (PBS-treated) ultracentrifugation-clarified cabbage lysate.
    Proteinase K, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 93/100, based on 2692 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs pngase f
    Forms of MIR2911 in cabbage extract. ( a ) Edible Plant Derived Exosome-like Nanoparticles (EPDENs) were purified from clarified cabbage lysate via ultracentrifugation. Levels of MIR2911 were quantified by qRT-PCR in various fractions: cabbage leaves liquefied in a blender (Liquefied Cabbage); Supernatant from 10,000 × g centrifugation (Slow Clarification); Supernatant from 150,000 × g ultracentrifugation (Ultra Clarification); EPDEN bands at 30% 45% interface (EPDEN band1) and 45% 60% interface (EPDEN band2) of sucrose gradient fractionation of total vesicles. ( b ) Size exclusion chromatography analysis of MIR2911 in ultracentrifugation-clarified cabbage lysate ( c ) qRT-PCR analysis of MIR2911 levels in proteinase K-treated or control (PBS-treated) ultracentrifugation-clarified cabbage lysate.
    Pngase F, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9623 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs lysis buffer
    Forms of MIR2911 in cabbage extract. ( a ) Edible Plant Derived Exosome-like Nanoparticles (EPDENs) were purified from clarified cabbage lysate via ultracentrifugation. Levels of MIR2911 were quantified by qRT-PCR in various fractions: cabbage leaves liquefied in a blender (Liquefied Cabbage); Supernatant from 10,000 × g centrifugation (Slow Clarification); Supernatant from 150,000 × g ultracentrifugation (Ultra Clarification); EPDEN bands at 30% 45% interface (EPDEN band1) and 45% 60% interface (EPDEN band2) of sucrose gradient fractionation of total vesicles. ( b ) Size exclusion chromatography analysis of MIR2911 in ultracentrifugation-clarified cabbage lysate ( c ) qRT-PCR analysis of MIR2911 levels in proteinase K-treated or control (PBS-treated) ultracentrifugation-clarified cabbage lysate.
    Lysis Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1883 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore formaldehyde solution
    Forms of MIR2911 in cabbage extract. ( a ) Edible Plant Derived Exosome-like Nanoparticles (EPDENs) were purified from clarified cabbage lysate via ultracentrifugation. Levels of MIR2911 were quantified by qRT-PCR in various fractions: cabbage leaves liquefied in a blender (Liquefied Cabbage); Supernatant from 10,000 × g centrifugation (Slow Clarification); Supernatant from 150,000 × g ultracentrifugation (Ultra Clarification); EPDEN bands at 30% 45% interface (EPDEN band1) and 45% 60% interface (EPDEN band2) of sucrose gradient fractionation of total vesicles. ( b ) Size exclusion chromatography analysis of MIR2911 in ultracentrifugation-clarified cabbage lysate ( c ) qRT-PCR analysis of MIR2911 levels in proteinase K-treated or control (PBS-treated) ultracentrifugation-clarified cabbage lysate.
    Formaldehyde Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 2274 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    IL-8 secretion induced by conditioned supernatants generated from Campylobacter -T84 cell coculture. (A) Polarized T84 cells were incubated for 24 h either with bacterium-free conditioned supernatants generated from the apical or basolateral supernatant of polarized T84 cells that had been inoculated with C. jejuni 81-176 for 4 h or with C. jejuni 81-176 alone cultured in the invasion medium for 4 h (bacterial). T84 cells were incubated with the bacterial culture medium in the apical chamber, with the apical conditioned supernatant in the apical chamber, or with the basolateral conditioned supernatant in the basolateral chamber. Polarized T84 cells inoculated apically with live C. jejuni 81-176 for 4 h and 24 h were used as controls. (B) The basolateral conditioned supernatant generated from C. jejuni 81-176-T84 cell coculture either was not pretreated (−) or was pretreated with either DNase I (10 U/ml) at 37°C for 2 h, polymyxin B (20 μg/ml) at 37°C for 30 min (PLXB), protease K (100 μg/ml) overnight followed by a 20-min incubation at 100°C (ProtK), or a 20-min incubation at 100°C without protease K (Boiling). Polarized T84 cells were incubated with the pretreated or untreated conditioned supernatants from wt C. jejuni 81-176-T84 cell coculture in the basolateral chamber at 37°C for 24 h. (C) Polarized T84 cells were treated basolaterally with a C. jejuni 81-176 DNA extract (25 μg/ml) (DNA) and DNase I-treated C. jejuni 81-176 DNA (DNA + DNase) at 37°C for 24 h. (D) Polarized T84 cells were incubated basolaterally with different concentrations of E. coli LPS in the presence or absence of 20 μg/ml PLXB. (E) Polarized T84 cells were incubated with conditioned supernatants from a coculture of T84 cells with wt C. jejuni 81-176 or its flaA , pflA , cdtB , or flaA cdtB mutant in the basolateral chamber at 37°C for 24 h. After different treatments, the apical (filled bars) and basolateral (open bars) media were collected. IL-8 concentrations were determined by ELISA. The data are means for three independent experiments; error bars, standard deviations. The data in panels B and E are expressed as the ratio of the IL-8 level induced by a treated basolateral conditioned supernatant to that induced by an untreated supernatant and as the ratio of the IL-8 level induced by a mutant basolateral conditioned supernatant to that induced by a wt supernatant. *, P

    Journal: Infection and Immunity

    Article Title: Campylobacter-Induced Interleukin-8 Secretion in Polarized Human Intestinal Epithelial Cells Requires Campylobacter-Secreted Cytolethal Distending Toxin- and Toll-Like Receptor-Mediated Activation of NF-?B ▿

    doi: 10.1128/IAI.01317-07

    Figure Lengend Snippet: IL-8 secretion induced by conditioned supernatants generated from Campylobacter -T84 cell coculture. (A) Polarized T84 cells were incubated for 24 h either with bacterium-free conditioned supernatants generated from the apical or basolateral supernatant of polarized T84 cells that had been inoculated with C. jejuni 81-176 for 4 h or with C. jejuni 81-176 alone cultured in the invasion medium for 4 h (bacterial). T84 cells were incubated with the bacterial culture medium in the apical chamber, with the apical conditioned supernatant in the apical chamber, or with the basolateral conditioned supernatant in the basolateral chamber. Polarized T84 cells inoculated apically with live C. jejuni 81-176 for 4 h and 24 h were used as controls. (B) The basolateral conditioned supernatant generated from C. jejuni 81-176-T84 cell coculture either was not pretreated (−) or was pretreated with either DNase I (10 U/ml) at 37°C for 2 h, polymyxin B (20 μg/ml) at 37°C for 30 min (PLXB), protease K (100 μg/ml) overnight followed by a 20-min incubation at 100°C (ProtK), or a 20-min incubation at 100°C without protease K (Boiling). Polarized T84 cells were incubated with the pretreated or untreated conditioned supernatants from wt C. jejuni 81-176-T84 cell coculture in the basolateral chamber at 37°C for 24 h. (C) Polarized T84 cells were treated basolaterally with a C. jejuni 81-176 DNA extract (25 μg/ml) (DNA) and DNase I-treated C. jejuni 81-176 DNA (DNA + DNase) at 37°C for 24 h. (D) Polarized T84 cells were incubated basolaterally with different concentrations of E. coli LPS in the presence or absence of 20 μg/ml PLXB. (E) Polarized T84 cells were incubated with conditioned supernatants from a coculture of T84 cells with wt C. jejuni 81-176 or its flaA , pflA , cdtB , or flaA cdtB mutant in the basolateral chamber at 37°C for 24 h. After different treatments, the apical (filled bars) and basolateral (open bars) media were collected. IL-8 concentrations were determined by ELISA. The data are means for three independent experiments; error bars, standard deviations. The data in panels B and E are expressed as the ratio of the IL-8 level induced by a treated basolateral conditioned supernatant to that induced by an untreated supernatant and as the ratio of the IL-8 level induced by a mutant basolateral conditioned supernatant to that induced by a wt supernatant. *, P

    Article Snippet: To inactivate heat-sensitive proteins, the collected conditioned supernatant was boiled for 20 min. To remove most of the protein factors, the conditioned supernatant was treated with protease K (100 μg/ml; Invitrogen) at 56°C overnight, followed by a 20-min incubation at 100°C to inactivate protease K. To remove bacterial DNA, the conditioned supernatant was treated with DNase I (10 U/ml; New England Biolabs, Ipswich, MA) at 37°C for 2 h. The treated supernatants were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12%) and agarose gel electrophoresis, confirming the removal of proteins by protease K and of bacterial DNA by DNase I (data not shown).

    Techniques: Generated, Incubation, Cell Culture, Mutagenesis, Enzyme-linked Immunosorbent Assay

    Forms of MIR2911 in cabbage extract. ( a ) Edible Plant Derived Exosome-like Nanoparticles (EPDENs) were purified from clarified cabbage lysate via ultracentrifugation. Levels of MIR2911 were quantified by qRT-PCR in various fractions: cabbage leaves liquefied in a blender (Liquefied Cabbage); Supernatant from 10,000 × g centrifugation (Slow Clarification); Supernatant from 150,000 × g ultracentrifugation (Ultra Clarification); EPDEN bands at 30% 45% interface (EPDEN band1) and 45% 60% interface (EPDEN band2) of sucrose gradient fractionation of total vesicles. ( b ) Size exclusion chromatography analysis of MIR2911 in ultracentrifugation-clarified cabbage lysate ( c ) qRT-PCR analysis of MIR2911 levels in proteinase K-treated or control (PBS-treated) ultracentrifugation-clarified cabbage lysate.

    Journal: Scientific Reports

    Article Title: Anomalous uptake and circulatory characteristics of the plant-based small RNA MIR2911

    doi: 10.1038/srep26834

    Figure Lengend Snippet: Forms of MIR2911 in cabbage extract. ( a ) Edible Plant Derived Exosome-like Nanoparticles (EPDENs) were purified from clarified cabbage lysate via ultracentrifugation. Levels of MIR2911 were quantified by qRT-PCR in various fractions: cabbage leaves liquefied in a blender (Liquefied Cabbage); Supernatant from 10,000 × g centrifugation (Slow Clarification); Supernatant from 150,000 × g ultracentrifugation (Ultra Clarification); EPDEN bands at 30% 45% interface (EPDEN band1) and 45% 60% interface (EPDEN band2) of sucrose gradient fractionation of total vesicles. ( b ) Size exclusion chromatography analysis of MIR2911 in ultracentrifugation-clarified cabbage lysate ( c ) qRT-PCR analysis of MIR2911 levels in proteinase K-treated or control (PBS-treated) ultracentrifugation-clarified cabbage lysate.

    Article Snippet: Proteinase K treatment Mouse sera or cabbage extract were mixed with proteinase K (P1807S, New England Biolabs, Ipswich, MA) at a final concentration of 1 mg/mL, or with the control PBS buffer and were incubated at 55 °C for 1 hour.

    Techniques: Derivative Assay, Purification, Quantitative RT-PCR, Centrifugation, Clarification Assay, Fractionation, Size-exclusion Chromatography

    Circulatory forms of MIR2911 in mice fed plant-containing diets. ( a ) Sera were obtained from mice fed either chamomile, honeysuckle, or chow diet. Exosomes were isolated from these mouse sera via ultracentrifugation protocol (UC) or PEG precipitation protocol (PEG). Levels of miR-16 and MIR2911 were quantified by qRT-PCR in the exosome pellets, or the supernatant fractions. ( b ) Size exclusion chromatography analysis of Let-7a, miR-16 and MIR2911 levels in control (untreated) serum samples from mice fed vegetable diets. ( c ) Time course analysis of miR-16 and MIR2911 levels in proteinase K-treated or control (untreated) serum samples from mice fed vegetable diets. ( d ) Size exclusion chromatography analysis of Let-7a, miR-16 and MIR2911 levels in proteinase K-treated serum samples from mice fed vegetable diets. Note: in Panel ( b ) and ( d ), the scale for Y axis for the 3 miRNA targets are set independently. E.g. for miR-16, the peak1 in panel ( b ) (arrow) represents similar concentration as the peak1 in panel ( d ) (arrow).

    Journal: Scientific Reports

    Article Title: Anomalous uptake and circulatory characteristics of the plant-based small RNA MIR2911

    doi: 10.1038/srep26834

    Figure Lengend Snippet: Circulatory forms of MIR2911 in mice fed plant-containing diets. ( a ) Sera were obtained from mice fed either chamomile, honeysuckle, or chow diet. Exosomes were isolated from these mouse sera via ultracentrifugation protocol (UC) or PEG precipitation protocol (PEG). Levels of miR-16 and MIR2911 were quantified by qRT-PCR in the exosome pellets, or the supernatant fractions. ( b ) Size exclusion chromatography analysis of Let-7a, miR-16 and MIR2911 levels in control (untreated) serum samples from mice fed vegetable diets. ( c ) Time course analysis of miR-16 and MIR2911 levels in proteinase K-treated or control (untreated) serum samples from mice fed vegetable diets. ( d ) Size exclusion chromatography analysis of Let-7a, miR-16 and MIR2911 levels in proteinase K-treated serum samples from mice fed vegetable diets. Note: in Panel ( b ) and ( d ), the scale for Y axis for the 3 miRNA targets are set independently. E.g. for miR-16, the peak1 in panel ( b ) (arrow) represents similar concentration as the peak1 in panel ( d ) (arrow).

    Article Snippet: Proteinase K treatment Mouse sera or cabbage extract were mixed with proteinase K (P1807S, New England Biolabs, Ipswich, MA) at a final concentration of 1 mg/mL, or with the control PBS buffer and were incubated at 55 °C for 1 hour.

    Techniques: Mouse Assay, Isolation, Quantitative RT-PCR, Size-exclusion Chromatography, Concentration Assay

    Single-neuron resolution RNA quantification. ( A ) A representative ExCel-processed (formaldehyde-fixed, β-mercaptoethanol-reduced, LabelX- and AcX-treated, hydrogel-embedded, Proteinase-K digested and re-embedded; as in Figure 1A–I, M, N ) L4 hermaphrodite animal labeled with anti-GFP (green), ExFISH-HCR against unc-25 mRNA transcript (red) and DAPI (not shown for image clarity). Boxes are manually selected ROIs enclosing single neurons that were identified based on stereotypical somatic location (via GFP and DAPI signal) and prior knowledge of unc-25 expression pattern (via ExFISH signal). Strain expressed tag-168p::GFP . Image is a max-intensity projection of a confocal stack acquired through the entire animal. Brightness and contrast settings: first set by the automatic adjustment function in Fiji, and then manually adjusted (raising the minimum-intensity threshold and lowering the maximum-intensity threshold) to improve contrast for cellular morphology and ExFISH puncta. Linear expansion factor: 3.2x. Scale bar: 10 μm. ( B ) Magnified view of the region of interest enclosing RMEV from A. Scale bar: 1 μm. ( C ) Same image as in B, with detected ExFISH-HCR spots (white circle) that correspond to unc-25 mRNA molecules. Spot detection was performed on the confocal stack by a 3D spot-finding algorithm (see Methods for details). ( D– F) mRNA molecule count of ( D ) unc-25 , ( E ) cat-2 and ( F ) tph-1 in each expressing neuron around the nerve ring, by applying the analytical workflow shown in A-C to separate groups of ExCel-processed animals singly labeled for each specified transcript target. Bar height, mean; error bars, standard deviation. Analyzed animals were between L2-L4 stages. n = 3–7 animals from 1 population, for each transcript-neuron combination. Values for neurons with n

    Journal: eLife

    Article Title: Expansion microscopy of C. elegans

    doi: 10.7554/eLife.46249

    Figure Lengend Snippet: Single-neuron resolution RNA quantification. ( A ) A representative ExCel-processed (formaldehyde-fixed, β-mercaptoethanol-reduced, LabelX- and AcX-treated, hydrogel-embedded, Proteinase-K digested and re-embedded; as in Figure 1A–I, M, N ) L4 hermaphrodite animal labeled with anti-GFP (green), ExFISH-HCR against unc-25 mRNA transcript (red) and DAPI (not shown for image clarity). Boxes are manually selected ROIs enclosing single neurons that were identified based on stereotypical somatic location (via GFP and DAPI signal) and prior knowledge of unc-25 expression pattern (via ExFISH signal). Strain expressed tag-168p::GFP . Image is a max-intensity projection of a confocal stack acquired through the entire animal. Brightness and contrast settings: first set by the automatic adjustment function in Fiji, and then manually adjusted (raising the minimum-intensity threshold and lowering the maximum-intensity threshold) to improve contrast for cellular morphology and ExFISH puncta. Linear expansion factor: 3.2x. Scale bar: 10 μm. ( B ) Magnified view of the region of interest enclosing RMEV from A. Scale bar: 1 μm. ( C ) Same image as in B, with detected ExFISH-HCR spots (white circle) that correspond to unc-25 mRNA molecules. Spot detection was performed on the confocal stack by a 3D spot-finding algorithm (see Methods for details). ( D– F) mRNA molecule count of ( D ) unc-25 , ( E ) cat-2 and ( F ) tph-1 in each expressing neuron around the nerve ring, by applying the analytical workflow shown in A-C to separate groups of ExCel-processed animals singly labeled for each specified transcript target. Bar height, mean; error bars, standard deviation. Analyzed animals were between L2-L4 stages. n = 3–7 animals from 1 population, for each transcript-neuron combination. Values for neurons with n

    Article Snippet: Standard ExCel: Proteinase K digestion Proteinase K (New England Biolabs, 800 U/mL stock) was diluted at 1:100 into non-expanding digestion buffer (50 mM Tris pH 8.0, 500 mM NaCl, 40 mM CaCl2 , 0.1% Triton X-100).

    Techniques: Labeling, Expressing, Standard Deviation

    ExCel enables simultaneous readout of fluorescent proteins, RNA, DNA location, and anatomical features. The pharyngeal region of a representative ExCel-processed (formaldehyde-fixed, β-mercaptoethanol-reduced, LabelX- and AcX-treated, hydrogel-embedded, Proteinase-K digested and re-embedded; as in Figure 1A–I, M, N ) L2 hermaphrodite animal, stained sequentially with ExFISH-HCR against the egfp mRNA, antibody against GFP, NHS ester of a fluorescent dye (Atto 647N NHS ester; against amines; for anatomical features) and DAPI (for DNA location), as schematized in Figure 1N–Q . ( A–D ) Single-channel images of each staining modality. ( E ) Merged composite image from combining A-D. Strain expressed tag-168p::GFP . Images are single-z-plane confocal micrographs. Brightness and contrast settings: each channel was first set by the automatic adjustment function in Fiji, and then manually adjusted (raising the minimum-intensity threshold and lowering the maximum-intensity threshold) to improve contrast. Linear expansion factor: 3.3x. Scale bars: 10 μm.

    Journal: eLife

    Article Title: Expansion microscopy of C. elegans

    doi: 10.7554/eLife.46249

    Figure Lengend Snippet: ExCel enables simultaneous readout of fluorescent proteins, RNA, DNA location, and anatomical features. The pharyngeal region of a representative ExCel-processed (formaldehyde-fixed, β-mercaptoethanol-reduced, LabelX- and AcX-treated, hydrogel-embedded, Proteinase-K digested and re-embedded; as in Figure 1A–I, M, N ) L2 hermaphrodite animal, stained sequentially with ExFISH-HCR against the egfp mRNA, antibody against GFP, NHS ester of a fluorescent dye (Atto 647N NHS ester; against amines; for anatomical features) and DAPI (for DNA location), as schematized in Figure 1N–Q . ( A–D ) Single-channel images of each staining modality. ( E ) Merged composite image from combining A-D. Strain expressed tag-168p::GFP . Images are single-z-plane confocal micrographs. Brightness and contrast settings: each channel was first set by the automatic adjustment function in Fiji, and then manually adjusted (raising the minimum-intensity threshold and lowering the maximum-intensity threshold) to improve contrast. Linear expansion factor: 3.3x. Scale bars: 10 μm.

    Article Snippet: Standard ExCel: Proteinase K digestion Proteinase K (New England Biolabs, 800 U/mL stock) was diluted at 1:100 into non-expanding digestion buffer (50 mM Tris pH 8.0, 500 mM NaCl, 40 mM CaCl2 , 0.1% Triton X-100).

    Techniques: Staining

    Screen of post-gelation treatments that confer tissue expandability and general stainability of epitopes. ( A ) Representative transillumination images of paraformaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, and hydrogel-embedded hermaphrodite animals, (left column) right after hydrogel embedding and prior to any hydrogel expansion, (middle column) after 1.9x-2.1x hydrogel expansion, by incubating the gelled sample in 1x PBS, or (right column) after 3.3–3.7x hydrogel expansion, by sequentially washing the gelled sample with 0.5x PBS, 0.1x PBS, and 0.01x PBS. After hydrogel embedding, gelled samples either are left in TNT buffer (top row; no treatment), processed with a 2 day 37°C Proteinase K digestion, as in the standard ExCel protocol (middle row), or processed with a 5 day 37°C collagenase type II digestion (bottom row). Transillumination images provide visualization to both the contour of the worm (traced under high digital magnification in black dotted lines, in cases where direct observation is difficult due to reduced tissue scattering after hydrogel expansion), and also the contour of the mold in the embedding hydrogel (traced in white dotted lines, in cases where direct observation is difficult to reduced gel-boundary scattering after hydrogel expansion). For each treatment, the expansion factor of the worm (measured as the length ratio of a worm in the pre-expansion and the post-3.5x-expansion (hydrogel) state) is normalized by the expansion factor of the embedding hydrogel, which results in a normalized expansion factor (abbreviated as nExF), to remove the variation on worm expansion factor due to inter-sample variation in the hydrogel expansion factor. For the no-treatment condition (top row) and the collagenase type II condition (bottom row), where the normalized expansion factors are markedly less than unity (0.40 and 0.66, respectively), the hydrogel-embedded worm tissue detaches from the surrounding hydrogel, due to tissue mechanical hindrances against expansion that are incompletely removed by the post-hydrogel-embedding treatment, and can be visualized by the extent of mismatch between the worm contour and the hydrogel-mold contour. Images are single-plane wide-field acquisitions. For post-2.0x- and 3.5x- images, in cases where uneven illumination from the bright-field light source strongly affects contour visualization, a band-pass filtering with the boundary of 3 and 30 pixels was performed with the Fiji function ‘Bandpass Filter’ to remove the illumination artifact, and to improve contour visualization. Brightness and contrast settings: first set by the automatic adjustment function in Fiji, and then manually adjusted (raising the minimum-intensity threshold and lowering the maximum-intensity threshold) to improve contrast for the boundaries of the worm and the mold. Scale bars: 300 μm in actual units (not converting to biological units here, since the two features (worm and hydrogel-mold) are associated with different expansion factors). ( B ) Representative images of the immunostaining of hydrogel-embedded animals in ( A ), via a panel of 5 primary antibodies with known patterns of staining. Due to spectral limitation, the five antibodies were separated into four spectrally separable channels (DyLight 405 for anti-GFP, Alexa 488 for anti-LMN1, Alexa 546 for anti-myotactin and anti-DLG1, Alexa 647 for anti-acetylated tubulin). An IHC score from 0 to 1 was manually assigned to each channel, based on the estimated signal-to-noise ratio of the expected pattern of staining, and thereby provides a rough quantification for the quality of immunostaining of each antibody (or pair of antibodies) following the specified post-hydrogel-embedding treatment. The strain used had pan-neuronal cytosolic expression of GFP ( tag-168p::GFP ). A few patterns of channel crosstalk, such as the anti-GFP signal observed in the anti-myotactin + anti-DLG1 channel, were observed but do not affect the scoring process, because the known patterns of staining for each of the five antibodies were spatially separable (GFP, pan-neuronal by promoter choice; LMN1 (lamin), nuclear; myotactin, periphery of pharyngeal muscle and beneath cuticle; DLG1 (disc large), adherens junctions that form characteristic thread-like patterns across the length of the worm; acetylated tubulin, touch-receptor neurons). Images are max-intensity projections of confocal stacks acquired through the entire animal. Brightness and contrast settings: individually set by the automatic adjustment function in Fiji. Linear expansion factors of the hydrogel: 1.9–2.1x (after immunostaining, the samples were left in 1x PBS and imaged in that state, without further expansion in deionized water; we decided to use this procedure here, because we observed that even at this partially expanded state, we could already evaluate whether the staining against protein targets yielded the expected patterns of localization, as demonstrated by the images in this panel, without the additional improvements in resolution that would result from further expansion of the samples). Linear expansion factors of the worm: no treatment, 1.1x; Proteinase K (standard ExCel), 1.9x, Collagenase Type II, 1.6x. Scale bars: left images, 50 μm (in biological units, i.e. post-expansion lengths are divided by the expansion factor of the worm). ( C ) Summary of the screen of 22 post-hydrogel-embedding treatments, each of which is characterized by (X axis) the post-treatment expandability of the worms, as quantified by the normalized expansion factor analysis as performed in A, and (Y axis) the post-treatment quality of immunohistochemistry, as quantified by the average of IHC scores across the four channels in the immunostaining assay as performed in B. Each dot represents a single treatment. See Methods for the protocol performed for each treatment. Treatments are grouped based on the nature of the protocol, and colored according to the group they belong to (legend). X- and Y- coordinates of each treatment represent the mean values of all animals analyzed in the expandability assay (which quantifies the normalized expansion factor, as in A) and the immunostaining assay (which quantifies the IHC score, as in B), respectively. Number of animals analyzed in the assay: expandability assay, 3–4 animals from 1 hydrogel sample; 4-channel immunostaining assay, 2–4 animals from 1 hydrogel sample, except for the papain treatment (1 animal). The condition displayed as MAP5 18-18-2 (heat denaturation in MAP5 buffer for 18 hr at 37°C, 18 hr at 70°C, and 2 hr at 95°C) is abbreviated as simply ‘MAP5’ in later figures. Source data of the measurements made in the expandability and the stainability assays are available in Figure 11—source data 1 . Measurements for the expandabiliy and stainability assays, whose population statistics are summarized in Figure 11C .

    Journal: eLife

    Article Title: Expansion microscopy of C. elegans

    doi: 10.7554/eLife.46249

    Figure Lengend Snippet: Screen of post-gelation treatments that confer tissue expandability and general stainability of epitopes. ( A ) Representative transillumination images of paraformaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, and hydrogel-embedded hermaphrodite animals, (left column) right after hydrogel embedding and prior to any hydrogel expansion, (middle column) after 1.9x-2.1x hydrogel expansion, by incubating the gelled sample in 1x PBS, or (right column) after 3.3–3.7x hydrogel expansion, by sequentially washing the gelled sample with 0.5x PBS, 0.1x PBS, and 0.01x PBS. After hydrogel embedding, gelled samples either are left in TNT buffer (top row; no treatment), processed with a 2 day 37°C Proteinase K digestion, as in the standard ExCel protocol (middle row), or processed with a 5 day 37°C collagenase type II digestion (bottom row). Transillumination images provide visualization to both the contour of the worm (traced under high digital magnification in black dotted lines, in cases where direct observation is difficult due to reduced tissue scattering after hydrogel expansion), and also the contour of the mold in the embedding hydrogel (traced in white dotted lines, in cases where direct observation is difficult to reduced gel-boundary scattering after hydrogel expansion). For each treatment, the expansion factor of the worm (measured as the length ratio of a worm in the pre-expansion and the post-3.5x-expansion (hydrogel) state) is normalized by the expansion factor of the embedding hydrogel, which results in a normalized expansion factor (abbreviated as nExF), to remove the variation on worm expansion factor due to inter-sample variation in the hydrogel expansion factor. For the no-treatment condition (top row) and the collagenase type II condition (bottom row), where the normalized expansion factors are markedly less than unity (0.40 and 0.66, respectively), the hydrogel-embedded worm tissue detaches from the surrounding hydrogel, due to tissue mechanical hindrances against expansion that are incompletely removed by the post-hydrogel-embedding treatment, and can be visualized by the extent of mismatch between the worm contour and the hydrogel-mold contour. Images are single-plane wide-field acquisitions. For post-2.0x- and 3.5x- images, in cases where uneven illumination from the bright-field light source strongly affects contour visualization, a band-pass filtering with the boundary of 3 and 30 pixels was performed with the Fiji function ‘Bandpass Filter’ to remove the illumination artifact, and to improve contour visualization. Brightness and contrast settings: first set by the automatic adjustment function in Fiji, and then manually adjusted (raising the minimum-intensity threshold and lowering the maximum-intensity threshold) to improve contrast for the boundaries of the worm and the mold. Scale bars: 300 μm in actual units (not converting to biological units here, since the two features (worm and hydrogel-mold) are associated with different expansion factors). ( B ) Representative images of the immunostaining of hydrogel-embedded animals in ( A ), via a panel of 5 primary antibodies with known patterns of staining. Due to spectral limitation, the five antibodies were separated into four spectrally separable channels (DyLight 405 for anti-GFP, Alexa 488 for anti-LMN1, Alexa 546 for anti-myotactin and anti-DLG1, Alexa 647 for anti-acetylated tubulin). An IHC score from 0 to 1 was manually assigned to each channel, based on the estimated signal-to-noise ratio of the expected pattern of staining, and thereby provides a rough quantification for the quality of immunostaining of each antibody (or pair of antibodies) following the specified post-hydrogel-embedding treatment. The strain used had pan-neuronal cytosolic expression of GFP ( tag-168p::GFP ). A few patterns of channel crosstalk, such as the anti-GFP signal observed in the anti-myotactin + anti-DLG1 channel, were observed but do not affect the scoring process, because the known patterns of staining for each of the five antibodies were spatially separable (GFP, pan-neuronal by promoter choice; LMN1 (lamin), nuclear; myotactin, periphery of pharyngeal muscle and beneath cuticle; DLG1 (disc large), adherens junctions that form characteristic thread-like patterns across the length of the worm; acetylated tubulin, touch-receptor neurons). Images are max-intensity projections of confocal stacks acquired through the entire animal. Brightness and contrast settings: individually set by the automatic adjustment function in Fiji. Linear expansion factors of the hydrogel: 1.9–2.1x (after immunostaining, the samples were left in 1x PBS and imaged in that state, without further expansion in deionized water; we decided to use this procedure here, because we observed that even at this partially expanded state, we could already evaluate whether the staining against protein targets yielded the expected patterns of localization, as demonstrated by the images in this panel, without the additional improvements in resolution that would result from further expansion of the samples). Linear expansion factors of the worm: no treatment, 1.1x; Proteinase K (standard ExCel), 1.9x, Collagenase Type II, 1.6x. Scale bars: left images, 50 μm (in biological units, i.e. post-expansion lengths are divided by the expansion factor of the worm). ( C ) Summary of the screen of 22 post-hydrogel-embedding treatments, each of which is characterized by (X axis) the post-treatment expandability of the worms, as quantified by the normalized expansion factor analysis as performed in A, and (Y axis) the post-treatment quality of immunohistochemistry, as quantified by the average of IHC scores across the four channels in the immunostaining assay as performed in B. Each dot represents a single treatment. See Methods for the protocol performed for each treatment. Treatments are grouped based on the nature of the protocol, and colored according to the group they belong to (legend). X- and Y- coordinates of each treatment represent the mean values of all animals analyzed in the expandability assay (which quantifies the normalized expansion factor, as in A) and the immunostaining assay (which quantifies the IHC score, as in B), respectively. Number of animals analyzed in the assay: expandability assay, 3–4 animals from 1 hydrogel sample; 4-channel immunostaining assay, 2–4 animals from 1 hydrogel sample, except for the papain treatment (1 animal). The condition displayed as MAP5 18-18-2 (heat denaturation in MAP5 buffer for 18 hr at 37°C, 18 hr at 70°C, and 2 hr at 95°C) is abbreviated as simply ‘MAP5’ in later figures. Source data of the measurements made in the expandability and the stainability assays are available in Figure 11—source data 1 . Measurements for the expandabiliy and stainability assays, whose population statistics are summarized in Figure 11C .

    Article Snippet: Standard ExCel: Proteinase K digestion Proteinase K (New England Biolabs, 800 U/mL stock) was diluted at 1:100 into non-expanding digestion buffer (50 mM Tris pH 8.0, 500 mM NaCl, 40 mM CaCl2 , 0.1% Triton X-100).

    Techniques: Immunostaining, Staining, Immunohistochemistry, Expressing

    Local distortion at the mouth region. Representative images of mouth regions of paraformaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, and hydrogel-embedded L1 – day 2 adult hermaphrodite C. elegans animals (as in Figure 1A–C, E–G ) before Proteinase K digestion, partial expansion to 1.8x, antibody staining, and expansion to 3.3x (‘pre-ExCel’) or after such treatments (‘post-ExCel’). Right images are magnified views of the boxed regions (centered on the mouth) in the left images. For each animal in this analysis (n = 20 animals from 2 separately processed populations), a score of distortion is manually assigned based on the perceived differences between the pre- and post- ExCel images of the mouth region. Images show animals whose distortion scores were ranked at the 5th, 25th, 50th, 75th and 95th percentile, to represent the distribution of the local distortion. The strain used had pan-neuronal cytosolic expression of GFP ( tag-168p::GFP ). Signals in the pre-ExCel images were from native GFP; signals in the post-ExCel images were from antibody staining against GFP. Images are max-intensity projections of confocal stacks acquired through the entire animal. Brightness and contrast settings: individually set by the automatic adjustment function in Fiji. Linear expansion factors: post-ExCel images, 3.2x. Scale bars: left images (i.e. images with boxed regions), 50 μm; right images (i.e. images without boxed regions), 10 μm.

    Journal: eLife

    Article Title: Expansion microscopy of C. elegans

    doi: 10.7554/eLife.46249

    Figure Lengend Snippet: Local distortion at the mouth region. Representative images of mouth regions of paraformaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, and hydrogel-embedded L1 – day 2 adult hermaphrodite C. elegans animals (as in Figure 1A–C, E–G ) before Proteinase K digestion, partial expansion to 1.8x, antibody staining, and expansion to 3.3x (‘pre-ExCel’) or after such treatments (‘post-ExCel’). Right images are magnified views of the boxed regions (centered on the mouth) in the left images. For each animal in this analysis (n = 20 animals from 2 separately processed populations), a score of distortion is manually assigned based on the perceived differences between the pre- and post- ExCel images of the mouth region. Images show animals whose distortion scores were ranked at the 5th, 25th, 50th, 75th and 95th percentile, to represent the distribution of the local distortion. The strain used had pan-neuronal cytosolic expression of GFP ( tag-168p::GFP ). Signals in the pre-ExCel images were from native GFP; signals in the post-ExCel images were from antibody staining against GFP. Images are max-intensity projections of confocal stacks acquired through the entire animal. Brightness and contrast settings: individually set by the automatic adjustment function in Fiji. Linear expansion factors: post-ExCel images, 3.2x. Scale bars: left images (i.e. images with boxed regions), 50 μm; right images (i.e. images without boxed regions), 10 μm.

    Article Snippet: Standard ExCel: Proteinase K digestion Proteinase K (New England Biolabs, 800 U/mL stock) was diluted at 1:100 into non-expanding digestion buffer (50 mM Tris pH 8.0, 500 mM NaCl, 40 mM CaCl2 , 0.1% Triton X-100).

    Techniques: Staining, Expressing

    Super-resolution imaging of synaptic proteins with ExCel. ( A ) Representative images of GFP-fused synaptic proteins RAB-3, SNB-1 and GLR-1 in paraformaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, and hydrogel-embedded (as in Figure 1A–C, E–G ) hermaphrodite animals, before Proteinase K digestion, partial expansion to 1.8x, antibody staining, and expansion to 3.8x (‘pre-ExCel’) or after such treatments (‘post-ExCel’). Middle images are magnified views of the boxed regions in the left images. Strains express GFP fusions of pre-synaptic proteins RAB-3 ( rab-3p::GFP::rab-3 ) or SNB-1 ( unc-25p::snb-1::GFP ), or post-synaptic protein GLR-1 ( glr-1p::glr-1::GFP ). Signals in the pre-ExCel images were from native GFP; signals in the post-ExCel images were from antibody staining against GFP. Images are max-intensity projections of confocal stacks acquired through the regions of interest. Brightness and contrast settings: left images, individually set by the automatic adjustment function in Fiji; center and right images, first set by the automatic adjustment function in Fiji, and then manually adjusted (raising the minimum-intensity threshold and lowering the maximum-intensity threshold) to improve contrast for the synaptic puncta. Linear expansion factors: post-ExCel images, 3.8x. Scale bars: left images, 20 μm; middle and right images, 1 μm. ( B ) Representative line intensity profiles of RAB-3::GFP along a section of the ventral nerve cord, from pre- (top) and post- (bottom) ExCel images acquired as in the top row of A (except that the last wash in deionized water is skipped, resulting in improved stability of antibody stained signal and 3.3x linear expansion, as discussed in Main Text). Fluorescent intensity values were linearly normalized to arbitrary units between 0 and 1. Expansion factors of the analyzed post-ExCel image: 3.3x. Asterisks, detected peaks. ( C ) Peak counts of the line intensity profiles of RAB-3::GFP along sections of ventral nerve cord or SAB axonal processes, pre- and post-ExCel, as plotted in B. Each dot represents a single line profile. Expansion factors of the analyzed post-ExCel images: 3.3x. Dashed line, unity; dotted line, linear fit. n = 16 line profiles from 7 animals in 2 separately processed populations. Source data of the line intensity profiles and their peak counts are available in Figure 6—source datas 1 and 2 , respectively. Line intensity profiles for all data points plotted in Figure 6C . Peak counts for all data points plotted in Figure 6C .

    Journal: eLife

    Article Title: Expansion microscopy of C. elegans

    doi: 10.7554/eLife.46249

    Figure Lengend Snippet: Super-resolution imaging of synaptic proteins with ExCel. ( A ) Representative images of GFP-fused synaptic proteins RAB-3, SNB-1 and GLR-1 in paraformaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, and hydrogel-embedded (as in Figure 1A–C, E–G ) hermaphrodite animals, before Proteinase K digestion, partial expansion to 1.8x, antibody staining, and expansion to 3.8x (‘pre-ExCel’) or after such treatments (‘post-ExCel’). Middle images are magnified views of the boxed regions in the left images. Strains express GFP fusions of pre-synaptic proteins RAB-3 ( rab-3p::GFP::rab-3 ) or SNB-1 ( unc-25p::snb-1::GFP ), or post-synaptic protein GLR-1 ( glr-1p::glr-1::GFP ). Signals in the pre-ExCel images were from native GFP; signals in the post-ExCel images were from antibody staining against GFP. Images are max-intensity projections of confocal stacks acquired through the regions of interest. Brightness and contrast settings: left images, individually set by the automatic adjustment function in Fiji; center and right images, first set by the automatic adjustment function in Fiji, and then manually adjusted (raising the minimum-intensity threshold and lowering the maximum-intensity threshold) to improve contrast for the synaptic puncta. Linear expansion factors: post-ExCel images, 3.8x. Scale bars: left images, 20 μm; middle and right images, 1 μm. ( B ) Representative line intensity profiles of RAB-3::GFP along a section of the ventral nerve cord, from pre- (top) and post- (bottom) ExCel images acquired as in the top row of A (except that the last wash in deionized water is skipped, resulting in improved stability of antibody stained signal and 3.3x linear expansion, as discussed in Main Text). Fluorescent intensity values were linearly normalized to arbitrary units between 0 and 1. Expansion factors of the analyzed post-ExCel image: 3.3x. Asterisks, detected peaks. ( C ) Peak counts of the line intensity profiles of RAB-3::GFP along sections of ventral nerve cord or SAB axonal processes, pre- and post-ExCel, as plotted in B. Each dot represents a single line profile. Expansion factors of the analyzed post-ExCel images: 3.3x. Dashed line, unity; dotted line, linear fit. n = 16 line profiles from 7 animals in 2 separately processed populations. Source data of the line intensity profiles and their peak counts are available in Figure 6—source datas 1 and 2 , respectively. Line intensity profiles for all data points plotted in Figure 6C . Peak counts for all data points plotted in Figure 6C .

    Article Snippet: Standard ExCel: Proteinase K digestion Proteinase K (New England Biolabs, 800 U/mL stock) was diluted at 1:100 into non-expanding digestion buffer (50 mM Tris pH 8.0, 500 mM NaCl, 40 mM CaCl2 , 0.1% Triton X-100).

    Techniques: Imaging, Staining

    ExCel enables antibody-mediated visualization of fluorescent proteins. ( A ) Representative images of immunohistochemistry against GFP in paraformaldehyde-fixed, β-mercaptoethanol-reduced (as in Figure 1A–C ) hermaphrodite animals, on which the antibody staining was performed without AcX treatment, hydrogel-embedding, Proteinase K digestion and partial expansion (‘pre-ExCel’) or with such treatments (‘post-ExCel’). The strain used had pan-neuronal cytosolic expression of GFP ( tag-168p::GFP ). Images throughout this figure are max-intensity projections of confocal stacks acquired through the entire animal. Brightness and contrast settings: DAPI (left) and the post-ExCel anti-GFP (lower center) images, individually set by the automatic adjustment function in Fiji; the pre-ExCel anti-GFP image (upper center), has the same settings as the post-ExCel anti-GFP image, to facilitate direct comparison. Linear expansion factor: lower images, 3.1x. Scale bars: 50 μm (in biological units, e.g. post-expansion lengths are divided by the expansion factor, used throughout this study unless otherwise noted). ( B ) Quantification of signal-to-background ratio for immunohistochemistry. Image shows a representative transgenic hermaphrodite animal ( tag-168p::GFP ), immunostained with anti-GFP post-ExCel, as in lower images of A. For quantitation, specimens were shrunk back to original size after antibody staining. Images at right are magnified views of the boxed regions at left (centered on the nerve ring and the upper body). Area masks (red) were generated to capture areas corresponding to neurons in the nerve ring region (representing the signal) and non-neuronal tissue (representing the background), using a semi-automated algorithm (see Methods for details). Scale bars: left image, 50 μm; right images, 10 μm. ( C ) Representative images of transgenic hermaphrodite C. elegans ( tag-168p::GFP ), immunostained with anti-GFP after various immunohistochemistry protocols (n = 11–17 animals from 3 separately fixed-and-stained populations for each protocol). For purposes of quantitation, the ExCel-processed sample was shrunk back to its original size after antibody staining. Lower panels are magnified views of the boxed regions (centered on the nerve ring) in upper panels. Brightness and contrast settings: each panel is individually set by the automatic adjustment function in Fiji. Scale bars: upper images, 100 μm; lower images, 20 μm. ( D ) Signal-to-background ratio of anti-GFP, computed as in B, for the immunohistochemistry methods performed on worms as in C, for various laser intensities (561 nm, since Alexa Fluor 546 was being imaged) and camera exposure times. Bars indicate mean + / - standard deviation. n = 15, 17, 15, 11 animals, from 3 separately fixed-and-stained populations for each protocol. Source data of the intensity measurements (signal and background), whose population statistics are summarized with the bar graph, are available in Figure 2—source data 1 . ( E ) Representative images for post-ExCel immunohistochemistry against different fluorescent proteins (n = 7 animals from 2 separately processed sets of animals for each strain) in hermaphrodite animals. The strains expressed one of the following gene constructs: tag-168p::GFP , rab-3p::mCherry , or rab-3p::NLS::TagRFP (NLS, nuclear localization sequence). Signals were from antibody staining (Alexa Fluor 546 for anti-GFP; Alexa Fluor 647 for anti-mCherry and anti-TagRFP). Brightness and contrast settings: individually set by the automatic adjustment function in Fiji. Linear expansion factors: 3.0–3.2x. Scale bars: 20 μm. ( F ) Signal-to-background ratio, plotted as in D (except with 561 and 647 nm lasers as appropriate, and using post-expansion images), of post-ExCel immunohistochemistry against fluorescent proteins from worms stained as in E. Linear expansion factors, 3.0–3.2x. n = 7 animals from 2 separately stained groups of animals for each strain. Source data of the intensity measurements (signal and background), whose population statistics are summarized with the bar graph, are available in Figure 2—source data 2 . Intensity measurements for the signal-to-background ratios shown in Figure 2D . Intensity measurements for the signal-to-background ratios shown in Figure 2F .

    Journal: eLife

    Article Title: Expansion microscopy of C. elegans

    doi: 10.7554/eLife.46249

    Figure Lengend Snippet: ExCel enables antibody-mediated visualization of fluorescent proteins. ( A ) Representative images of immunohistochemistry against GFP in paraformaldehyde-fixed, β-mercaptoethanol-reduced (as in Figure 1A–C ) hermaphrodite animals, on which the antibody staining was performed without AcX treatment, hydrogel-embedding, Proteinase K digestion and partial expansion (‘pre-ExCel’) or with such treatments (‘post-ExCel’). The strain used had pan-neuronal cytosolic expression of GFP ( tag-168p::GFP ). Images throughout this figure are max-intensity projections of confocal stacks acquired through the entire animal. Brightness and contrast settings: DAPI (left) and the post-ExCel anti-GFP (lower center) images, individually set by the automatic adjustment function in Fiji; the pre-ExCel anti-GFP image (upper center), has the same settings as the post-ExCel anti-GFP image, to facilitate direct comparison. Linear expansion factor: lower images, 3.1x. Scale bars: 50 μm (in biological units, e.g. post-expansion lengths are divided by the expansion factor, used throughout this study unless otherwise noted). ( B ) Quantification of signal-to-background ratio for immunohistochemistry. Image shows a representative transgenic hermaphrodite animal ( tag-168p::GFP ), immunostained with anti-GFP post-ExCel, as in lower images of A. For quantitation, specimens were shrunk back to original size after antibody staining. Images at right are magnified views of the boxed regions at left (centered on the nerve ring and the upper body). Area masks (red) were generated to capture areas corresponding to neurons in the nerve ring region (representing the signal) and non-neuronal tissue (representing the background), using a semi-automated algorithm (see Methods for details). Scale bars: left image, 50 μm; right images, 10 μm. ( C ) Representative images of transgenic hermaphrodite C. elegans ( tag-168p::GFP ), immunostained with anti-GFP after various immunohistochemistry protocols (n = 11–17 animals from 3 separately fixed-and-stained populations for each protocol). For purposes of quantitation, the ExCel-processed sample was shrunk back to its original size after antibody staining. Lower panels are magnified views of the boxed regions (centered on the nerve ring) in upper panels. Brightness and contrast settings: each panel is individually set by the automatic adjustment function in Fiji. Scale bars: upper images, 100 μm; lower images, 20 μm. ( D ) Signal-to-background ratio of anti-GFP, computed as in B, for the immunohistochemistry methods performed on worms as in C, for various laser intensities (561 nm, since Alexa Fluor 546 was being imaged) and camera exposure times. Bars indicate mean + / - standard deviation. n = 15, 17, 15, 11 animals, from 3 separately fixed-and-stained populations for each protocol. Source data of the intensity measurements (signal and background), whose population statistics are summarized with the bar graph, are available in Figure 2—source data 1 . ( E ) Representative images for post-ExCel immunohistochemistry against different fluorescent proteins (n = 7 animals from 2 separately processed sets of animals for each strain) in hermaphrodite animals. The strains expressed one of the following gene constructs: tag-168p::GFP , rab-3p::mCherry , or rab-3p::NLS::TagRFP (NLS, nuclear localization sequence). Signals were from antibody staining (Alexa Fluor 546 for anti-GFP; Alexa Fluor 647 for anti-mCherry and anti-TagRFP). Brightness and contrast settings: individually set by the automatic adjustment function in Fiji. Linear expansion factors: 3.0–3.2x. Scale bars: 20 μm. ( F ) Signal-to-background ratio, plotted as in D (except with 561 and 647 nm lasers as appropriate, and using post-expansion images), of post-ExCel immunohistochemistry against fluorescent proteins from worms stained as in E. Linear expansion factors, 3.0–3.2x. n = 7 animals from 2 separately stained groups of animals for each strain. Source data of the intensity measurements (signal and background), whose population statistics are summarized with the bar graph, are available in Figure 2—source data 2 . Intensity measurements for the signal-to-background ratios shown in Figure 2D . Intensity measurements for the signal-to-background ratios shown in Figure 2F .

    Article Snippet: Standard ExCel: Proteinase K digestion Proteinase K (New England Biolabs, 800 U/mL stock) was diluted at 1:100 into non-expanding digestion buffer (50 mM Tris pH 8.0, 500 mM NaCl, 40 mM CaCl2 , 0.1% Triton X-100).

    Techniques: Immunohistochemistry, Staining, Expressing, Transgenic Assay, Quantitation Assay, Generated, Standard Deviation, Construct, Sequencing

    Super-resolution imaging of electrical synapses with ExCel. Representative images of TagRFP-fused innexin protein CHE-7 in a paraformaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, and hydrogel-embedded (as in Figure 1A–C, E–G ) L4 hermaphrodite animal, before Proteinase K digestion, partial expansion to 1.8x, antibody staining, and expansion to 3.8x (‘pre-ExCel’) or after such treatments (‘post-ExCel’). Strain expresses innexin protein CHE-7 that is fused to TagRFP at its endogenous locus, via CRISPR-Cas9-mediated homologous recombination. ( A ) Pharyngeal region of the animal. The nerve ring is marked by the dotted box and shown in magnified views in the top panels of B. ( B ) Top panels, nerve ring of the animal, as marked in the dotted box in A. Lower panels, magnified views of the dotted regions in the top panels. Signals in the pre-ExCel images were from native TagRFP; signals in the post-ExCel images were from antibody staining against TagRFP. Images are max-intensity projections of confocal stacks acquired through the depth of the entire animal. Brightness and contrast settings: first set by the automatic adjustment function in Fiji, and then manually adjusted (raising the minimum-intensity threshold and lowering the maximum-intensity threshold) to improve contrast for the synaptic puncta. Linear expansion factors: post-ExCel images, 4.0x. Scale bars: ( A ) 50 μm; ( B ) top panels, 5 μm; bottom panels, 1 μm.

    Journal: eLife

    Article Title: Expansion microscopy of C. elegans

    doi: 10.7554/eLife.46249

    Figure Lengend Snippet: Super-resolution imaging of electrical synapses with ExCel. Representative images of TagRFP-fused innexin protein CHE-7 in a paraformaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, and hydrogel-embedded (as in Figure 1A–C, E–G ) L4 hermaphrodite animal, before Proteinase K digestion, partial expansion to 1.8x, antibody staining, and expansion to 3.8x (‘pre-ExCel’) or after such treatments (‘post-ExCel’). Strain expresses innexin protein CHE-7 that is fused to TagRFP at its endogenous locus, via CRISPR-Cas9-mediated homologous recombination. ( A ) Pharyngeal region of the animal. The nerve ring is marked by the dotted box and shown in magnified views in the top panels of B. ( B ) Top panels, nerve ring of the animal, as marked in the dotted box in A. Lower panels, magnified views of the dotted regions in the top panels. Signals in the pre-ExCel images were from native TagRFP; signals in the post-ExCel images were from antibody staining against TagRFP. Images are max-intensity projections of confocal stacks acquired through the depth of the entire animal. Brightness and contrast settings: first set by the automatic adjustment function in Fiji, and then manually adjusted (raising the minimum-intensity threshold and lowering the maximum-intensity threshold) to improve contrast for the synaptic puncta. Linear expansion factors: post-ExCel images, 4.0x. Scale bars: ( A ) 50 μm; ( B ) top panels, 5 μm; bottom panels, 1 μm.

    Article Snippet: Standard ExCel: Proteinase K digestion Proteinase K (New England Biolabs, 800 U/mL stock) was diluted at 1:100 into non-expanding digestion buffer (50 mM Tris pH 8.0, 500 mM NaCl, 40 mM CaCl2 , 0.1% Triton X-100).

    Techniques: Imaging, Staining, CRISPR, Homologous Recombination

    Workflow for iterative expansion of C. elegans (iExCel) sample processing. A method for iteratively expanding cuticle-enclosed intact C. elegans , for a final linear expansion factor of ~20x. Sample processing prior to Panel A is identical to the workflow for the standard ExCel protocol without ExFISH (as shown in blue arrows in Figure 1 ) until, and including, the post-Proteinase-K partial expansion step ( Figure 1A–C, E–G ). The linear expansion factor of the hydrogel-specimen composite is shown in parentheses. ( A–I ) Steps of the protocol, with the bold text indicating the title of the step. ( A ) Specimens are partially expanded from a linear expansion factor of 1.0x to 1.8x, with the same protocol as shown in Figure 1I . ( B ) Specimens are immunostained first with primary antibodies against fluorescent proteins in 5x SSCT overnight at 4°C, and then with secondary antibodies that have been conjugated to a 24-base DNA oligonucleotide, in DNA-conjugated Antibody Staining Buffer (2x SSC, 2% (w/v) dextran sulfate, 1 mg/mL yeast tRNA, 5%(v/v) normal donkey serum, 0.1% Triton X-100) overnight at 4°C. The DNA oligo is conjugated to the antibody at the 3’ end, and contains a gel anchorable group at the 5’ end. ( C ) Specimens are expanded from a linear expansion factor of 1.8x to 3.8x, with the same protocol as shown in Figure 1M . ( D ) Specimens are re-embedded into another non-expandable hydrogel (‘Gel #2’) to lock up its size at the expanded state, as shown in Figure 1N , except that the monomer solution is replaced by DATD-crosslinked re-embedding monomer solution (10% acrylamide, 1% N,N'-diallyl-tartardiamide (DATD), 0.05% TEMED, 0.05% APS), which results in a hydrogel that can be later disintegrated via crosslinker cleavage, to allow full expansion of the final expandable gel. The DATD-crosslinked re-embedding monomer solution contains a charged molecule APS. Therefore, the linear expansion factor slightly drops from 3.8x to 3.6x during this step. During hydrogel polymerization, the DNA oligo on the antibody, which contains a gel-anchorable group, is covalently anchored to the second hydrogel network (orange grids). ( E ) Specimens are incubated with a 100-base DNA oligonucleotide (‘Linker’), which hybridizes to the 24-base DNA oligo on the secondary antibodies, and which contains a gel anchorable group on its 5’ end, in iExCel hybridization buffer (4x SSC, 20% (v/v) formamide) overnight at RT. ( F ) Specimens are re-embedded into another expandable hydrogel (‘Gel #3’), by incubating the specimens in activated Gel #3 monomer solution (1x PBS pH 7.4, 7.5% (w/w) sodium acrylate, 2.5% (w/w) acrylamide, 0.15% (w/w) N,N’-methylene-bis-acrylamide, 2M NaCl, 0.015% 4-hydroxy-TEMPO, 0.2% TEMED, 0.2% APS) for 50 min at 4°C, transferring the specimens into a gelation chamber, and incubating the chamber for 2 hr at 37°C. During polymerization, the linker DNA oligo, which contains a gel-anchorable group, is covalently anchored to the hydrogel network of the third hydrogel (magenta grids). ( G ) Specimens are treated with DATD cleaving solution (20 mM sodium meta-periodate in 1x PBS, pH 5.5) for 30 min at RT, to chemically disintegrate the first and the second hydrogels, which contain a periodate-cleavable crosslinker N,N'-diallyl-tartardiamide (DATD), while sparing the third hydrogel, which contains a periodate-resistant crosslinker N,N’-methylene-bis-acrylamide (bis). ( H ) Specimens are incubated with a fluorophore-conjugated 15-base locked nucleic acid (LNA) oligonucleotide, which hybridizes to the 100-base linker DNA oligo at four locations, in iExCel hybridization buffer (4x SSC, 20% (v/v) formamide) overnight at RT. ( I ) Specimens are expanded to a linear expansion factor of ~20x, with three washes in deionized water. After expansion, specimens are ready for imaging.

    Journal: eLife

    Article Title: Expansion microscopy of C. elegans

    doi: 10.7554/eLife.46249

    Figure Lengend Snippet: Workflow for iterative expansion of C. elegans (iExCel) sample processing. A method for iteratively expanding cuticle-enclosed intact C. elegans , for a final linear expansion factor of ~20x. Sample processing prior to Panel A is identical to the workflow for the standard ExCel protocol without ExFISH (as shown in blue arrows in Figure 1 ) until, and including, the post-Proteinase-K partial expansion step ( Figure 1A–C, E–G ). The linear expansion factor of the hydrogel-specimen composite is shown in parentheses. ( A–I ) Steps of the protocol, with the bold text indicating the title of the step. ( A ) Specimens are partially expanded from a linear expansion factor of 1.0x to 1.8x, with the same protocol as shown in Figure 1I . ( B ) Specimens are immunostained first with primary antibodies against fluorescent proteins in 5x SSCT overnight at 4°C, and then with secondary antibodies that have been conjugated to a 24-base DNA oligonucleotide, in DNA-conjugated Antibody Staining Buffer (2x SSC, 2% (w/v) dextran sulfate, 1 mg/mL yeast tRNA, 5%(v/v) normal donkey serum, 0.1% Triton X-100) overnight at 4°C. The DNA oligo is conjugated to the antibody at the 3’ end, and contains a gel anchorable group at the 5’ end. ( C ) Specimens are expanded from a linear expansion factor of 1.8x to 3.8x, with the same protocol as shown in Figure 1M . ( D ) Specimens are re-embedded into another non-expandable hydrogel (‘Gel #2’) to lock up its size at the expanded state, as shown in Figure 1N , except that the monomer solution is replaced by DATD-crosslinked re-embedding monomer solution (10% acrylamide, 1% N,N'-diallyl-tartardiamide (DATD), 0.05% TEMED, 0.05% APS), which results in a hydrogel that can be later disintegrated via crosslinker cleavage, to allow full expansion of the final expandable gel. The DATD-crosslinked re-embedding monomer solution contains a charged molecule APS. Therefore, the linear expansion factor slightly drops from 3.8x to 3.6x during this step. During hydrogel polymerization, the DNA oligo on the antibody, which contains a gel-anchorable group, is covalently anchored to the second hydrogel network (orange grids). ( E ) Specimens are incubated with a 100-base DNA oligonucleotide (‘Linker’), which hybridizes to the 24-base DNA oligo on the secondary antibodies, and which contains a gel anchorable group on its 5’ end, in iExCel hybridization buffer (4x SSC, 20% (v/v) formamide) overnight at RT. ( F ) Specimens are re-embedded into another expandable hydrogel (‘Gel #3’), by incubating the specimens in activated Gel #3 monomer solution (1x PBS pH 7.4, 7.5% (w/w) sodium acrylate, 2.5% (w/w) acrylamide, 0.15% (w/w) N,N’-methylene-bis-acrylamide, 2M NaCl, 0.015% 4-hydroxy-TEMPO, 0.2% TEMED, 0.2% APS) for 50 min at 4°C, transferring the specimens into a gelation chamber, and incubating the chamber for 2 hr at 37°C. During polymerization, the linker DNA oligo, which contains a gel-anchorable group, is covalently anchored to the hydrogel network of the third hydrogel (magenta grids). ( G ) Specimens are treated with DATD cleaving solution (20 mM sodium meta-periodate in 1x PBS, pH 5.5) for 30 min at RT, to chemically disintegrate the first and the second hydrogels, which contain a periodate-cleavable crosslinker N,N'-diallyl-tartardiamide (DATD), while sparing the third hydrogel, which contains a periodate-resistant crosslinker N,N’-methylene-bis-acrylamide (bis). ( H ) Specimens are incubated with a fluorophore-conjugated 15-base locked nucleic acid (LNA) oligonucleotide, which hybridizes to the 100-base linker DNA oligo at four locations, in iExCel hybridization buffer (4x SSC, 20% (v/v) formamide) overnight at RT. ( I ) Specimens are expanded to a linear expansion factor of ~20x, with three washes in deionized water. After expansion, specimens are ready for imaging.

    Article Snippet: Standard ExCel: Proteinase K digestion Proteinase K (New England Biolabs, 800 U/mL stock) was diluted at 1:100 into non-expanding digestion buffer (50 mM Tris pH 8.0, 500 mM NaCl, 40 mM CaCl2 , 0.1% Triton X-100).

    Techniques: Staining, Incubation, Hybridization, Transferring, Imaging

    RNA detection in neurons. ExCel-processed (formaldehyde-fixed, β-mercaptoethanol-reduced, LabelX- and AcX-treated, hydrogel-embedded, Proteinase-K digested and re-embedded; as in Figure 1A–I, M, N ) hermaphrodite animals labeled with antibody staining against GFP (green) and ExFISH-HCR (magenta) against the following RNA transcripts: ( A ) egfp (as a positive control), ( B ) mouse parvalbumin (no known ortholog in C. elegans ; as a negative control), ( C ) unc-25 , a GABAergic neuronal marker, ( D ) cat-2 , a dopaminergic neuronal marker, ( E ) tph-1 , a serotoninergic neuronal marker, ( F ) cho-1 , a cholinergic neuronal marker, ( G ) eat-4 , a glutamatergic neuronal marker, ( H ) rab-3 , a pre-synaptic protein with pan-neuronal expression. Right images are magnified views of the boxed regions in the left images. Strain expressed tag-168p::GFP . Images are max-intensity projections of confocal stacks acquired through the entire animal (left images) or just the expressing cells (right images). Brightness and contrast settings: first set by the automatic adjustment function in Fiji, and then manually adjusted (raising the minimum-intensity threshold and lowering the maximum-intensity threshold) to improve contrast for cellular morphology and ExFISH puncta. Selection of displayed image: ( A–B ) both localization and density are representative, ( C–H ) localization is representative; density is close to exemplar, because the authors selected images from animals that have relatively strong expression levels, within the 3–5 animals imaged per transcript target, to facilitate visualization of the expressing cells. Nearly all of the selected images are from L2-L4 stage larvae, which have generally greater expression levels than adults, for the transcript targets that we investigated in these panels. Linear expansion factors: 3.4–3.6x. Scale bars: left images, 20 μm; right images, 5 μm.

    Journal: eLife

    Article Title: Expansion microscopy of C. elegans

    doi: 10.7554/eLife.46249

    Figure Lengend Snippet: RNA detection in neurons. ExCel-processed (formaldehyde-fixed, β-mercaptoethanol-reduced, LabelX- and AcX-treated, hydrogel-embedded, Proteinase-K digested and re-embedded; as in Figure 1A–I, M, N ) hermaphrodite animals labeled with antibody staining against GFP (green) and ExFISH-HCR (magenta) against the following RNA transcripts: ( A ) egfp (as a positive control), ( B ) mouse parvalbumin (no known ortholog in C. elegans ; as a negative control), ( C ) unc-25 , a GABAergic neuronal marker, ( D ) cat-2 , a dopaminergic neuronal marker, ( E ) tph-1 , a serotoninergic neuronal marker, ( F ) cho-1 , a cholinergic neuronal marker, ( G ) eat-4 , a glutamatergic neuronal marker, ( H ) rab-3 , a pre-synaptic protein with pan-neuronal expression. Right images are magnified views of the boxed regions in the left images. Strain expressed tag-168p::GFP . Images are max-intensity projections of confocal stacks acquired through the entire animal (left images) or just the expressing cells (right images). Brightness and contrast settings: first set by the automatic adjustment function in Fiji, and then manually adjusted (raising the minimum-intensity threshold and lowering the maximum-intensity threshold) to improve contrast for cellular morphology and ExFISH puncta. Selection of displayed image: ( A–B ) both localization and density are representative, ( C–H ) localization is representative; density is close to exemplar, because the authors selected images from animals that have relatively strong expression levels, within the 3–5 animals imaged per transcript target, to facilitate visualization of the expressing cells. Nearly all of the selected images are from L2-L4 stage larvae, which have generally greater expression levels than adults, for the transcript targets that we investigated in these panels. Linear expansion factors: 3.4–3.6x. Scale bars: left images, 20 μm; right images, 5 μm.

    Article Snippet: Standard ExCel: Proteinase K digestion Proteinase K (New England Biolabs, 800 U/mL stock) was diluted at 1:100 into non-expanding digestion buffer (50 mM Tris pH 8.0, 500 mM NaCl, 40 mM CaCl2 , 0.1% Triton X-100).

    Techniques: RNA Detection, Labeling, Staining, Positive Control, Negative Control, Marker, Expressing, Selection

    RNA detection in neurons, at sub-cellular resolution. Representative images of ExCel-processed (formaldehyde-fixed, β-mercaptoethanol-reduced, LabelX- and AcX-treated, hydrogel-embedded, Proteinase-K digested and re-embedded; as in Figure 1A–I, M, N ) hermaphrodite animals labeled with anti-GFP (green), DAPI (blue) and ExFISH-HCR (red) against specified mRNA transcripts (red text). Strain expressed tag-168p::GFP . Brightness and contrast settings: first set by the automatic adjustment function in Fiji, and then manually adjusted (raising the minimum-intensity threshold and lowering the maximum-intensity threshold) to improve contrast for cellular morphology and ExFISH puncta. Linear expansion factors: 3.1–3.3x. Scale bars: 5 μm. ( A ) Sub-cellular localization of mRNA transcripts in identified single neurons. White text indicates the identity of the displayed neuron, which was determined based on stereotypical somatic location (via GFP signal) and prior knowledge of the expression patterns (via ExFISH signal). Images are max-intensity projections over 5 z-planes (with step size of 0.4 μm, in absolute distance, i.e. post-expansion distance) centered (in z-dimension) at the centerline of the imaged cell. ( B ) Localization of egfp mRNA transcript in the head region of an L2 larval animal. White arrowhead, nerve ring.

    Journal: eLife

    Article Title: Expansion microscopy of C. elegans

    doi: 10.7554/eLife.46249

    Figure Lengend Snippet: RNA detection in neurons, at sub-cellular resolution. Representative images of ExCel-processed (formaldehyde-fixed, β-mercaptoethanol-reduced, LabelX- and AcX-treated, hydrogel-embedded, Proteinase-K digested and re-embedded; as in Figure 1A–I, M, N ) hermaphrodite animals labeled with anti-GFP (green), DAPI (blue) and ExFISH-HCR (red) against specified mRNA transcripts (red text). Strain expressed tag-168p::GFP . Brightness and contrast settings: first set by the automatic adjustment function in Fiji, and then manually adjusted (raising the minimum-intensity threshold and lowering the maximum-intensity threshold) to improve contrast for cellular morphology and ExFISH puncta. Linear expansion factors: 3.1–3.3x. Scale bars: 5 μm. ( A ) Sub-cellular localization of mRNA transcripts in identified single neurons. White text indicates the identity of the displayed neuron, which was determined based on stereotypical somatic location (via GFP signal) and prior knowledge of the expression patterns (via ExFISH signal). Images are max-intensity projections over 5 z-planes (with step size of 0.4 μm, in absolute distance, i.e. post-expansion distance) centered (in z-dimension) at the centerline of the imaged cell. ( B ) Localization of egfp mRNA transcript in the head region of an L2 larval animal. White arrowhead, nerve ring.

    Article Snippet: Standard ExCel: Proteinase K digestion Proteinase K (New England Biolabs, 800 U/mL stock) was diluted at 1:100 into non-expanding digestion buffer (50 mM Tris pH 8.0, 500 mM NaCl, 40 mM CaCl2 , 0.1% Triton X-100).

    Techniques: RNA Detection, Labeling, Expressing

    Post-ExCel NHS-ester staining reveals anatomical structures. Representative images of ( A ) pharyngeal region, ( B ) intestinal tissue and ( C ) gonad tissue of ExCel-processed (formaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, hydrogel-embedded, Proteinase-K digested, partially expanded; as in Figure 1A–C, E–G ) L3-L4 hermaphrodite animals, stained with Atto 647N NHS ester, which is an NHS ester of a fluorescent dye (as in Figure 1K ), and then expanded in deionized water (as in Figure 1L ). The strain used had pan-neuronal expression of RAB-3::GFP ( rab-3p::GFP::rab-3 ); not visualized in this specific set of images. Images are confocal micrographs at a single z-plane. Brightness and contrast settings: first set by the automatic adjustment function in Fiji, and then manually adjusted (raising the minimum-intensity threshold and lowering the maximum-intensity threshold) to improve contrast. Linear expansion factor: 4.1–4.2x. Scale bars: 10 μm.

    Journal: eLife

    Article Title: Expansion microscopy of C. elegans

    doi: 10.7554/eLife.46249

    Figure Lengend Snippet: Post-ExCel NHS-ester staining reveals anatomical structures. Representative images of ( A ) pharyngeal region, ( B ) intestinal tissue and ( C ) gonad tissue of ExCel-processed (formaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, hydrogel-embedded, Proteinase-K digested, partially expanded; as in Figure 1A–C, E–G ) L3-L4 hermaphrodite animals, stained with Atto 647N NHS ester, which is an NHS ester of a fluorescent dye (as in Figure 1K ), and then expanded in deionized water (as in Figure 1L ). The strain used had pan-neuronal expression of RAB-3::GFP ( rab-3p::GFP::rab-3 ); not visualized in this specific set of images. Images are confocal micrographs at a single z-plane. Brightness and contrast settings: first set by the automatic adjustment function in Fiji, and then manually adjusted (raising the minimum-intensity threshold and lowering the maximum-intensity threshold) to improve contrast. Linear expansion factor: 4.1–4.2x. Scale bars: 10 μm.

    Article Snippet: Standard ExCel: Proteinase K digestion Proteinase K (New England Biolabs, 800 U/mL stock) was diluted at 1:100 into non-expanding digestion buffer (50 mM Tris pH 8.0, 500 mM NaCl, 40 mM CaCl2 , 0.1% Triton X-100).

    Techniques: Staining, Expressing

    Local distortion at the gonad region of day 1 – day 2 adult hermaphrodites. Representative images of adult gonad regions of paraformaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, and hydrogel-embedded (as in Figure 1A–C, E–G ) hermaphrodite animals before Proteinase K digestion, partial expansion to 1.8x, antibody staining, and expansion to 3.3x (‘pre-ExCel’) or after such treatments (‘post-ExCel’). Right images are magnified views of the boxed regions (centered on the vulvae) in the left images. For each adult in this analysis (n = 20 animals from 2 separately processed populations), a score of distortion is manually assigned based on the perceived differences between the pre- and post- ExCel images of the gonad region. Images show adult animals whose distortion scores were ranked at the 5th, 25th, 50th, 75th and 95th percentile, to represent the distribution of the local distortion. The strain used had pan-neuronal cytosolic expression of GFP ( tag-168p::GFP ). Signals in the pre-ExCel images were from native GFP; signals in the post-ExCel images were from antibody staining against GFP. Images are max-intensity projections of confocal stacks acquired through the entire animal. Brightness and contrast settings: individually set by the automatic adjustment function in Fiji. Linear expansion factors: post-ExCel images, 3.2x. Scale bars: left images (i.e. images with boxed regions), 100 μm; right images (i.e. images without boxed regions), 50 μm.

    Journal: eLife

    Article Title: Expansion microscopy of C. elegans

    doi: 10.7554/eLife.46249

    Figure Lengend Snippet: Local distortion at the gonad region of day 1 – day 2 adult hermaphrodites. Representative images of adult gonad regions of paraformaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, and hydrogel-embedded (as in Figure 1A–C, E–G ) hermaphrodite animals before Proteinase K digestion, partial expansion to 1.8x, antibody staining, and expansion to 3.3x (‘pre-ExCel’) or after such treatments (‘post-ExCel’). Right images are magnified views of the boxed regions (centered on the vulvae) in the left images. For each adult in this analysis (n = 20 animals from 2 separately processed populations), a score of distortion is manually assigned based on the perceived differences between the pre- and post- ExCel images of the gonad region. Images show adult animals whose distortion scores were ranked at the 5th, 25th, 50th, 75th and 95th percentile, to represent the distribution of the local distortion. The strain used had pan-neuronal cytosolic expression of GFP ( tag-168p::GFP ). Signals in the pre-ExCel images were from native GFP; signals in the post-ExCel images were from antibody staining against GFP. Images are max-intensity projections of confocal stacks acquired through the entire animal. Brightness and contrast settings: individually set by the automatic adjustment function in Fiji. Linear expansion factors: post-ExCel images, 3.2x. Scale bars: left images (i.e. images with boxed regions), 100 μm; right images (i.e. images without boxed regions), 50 μm.

    Article Snippet: Standard ExCel: Proteinase K digestion Proteinase K (New England Biolabs, 800 U/mL stock) was diluted at 1:100 into non-expanding digestion buffer (50 mM Tris pH 8.0, 500 mM NaCl, 40 mM CaCl2 , 0.1% Triton X-100).

    Techniques: Staining, Expressing