complement factor h mouse hycult biotech  (Hycult Biotech)


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    Hycult Biotech complement factor h mouse hycult biotech
    Complement Factor H Mouse Hycult Biotech, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 91 stars, based on 1 article reviews
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    complement factor h mouse hycult biotech  (Hycult Biotech)


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    Hycult Biotech complement factor h mouse hycult biotech
    Complement Factor H Mouse Hycult Biotech, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/complement factor h mouse hycult biotech/product/Hycult Biotech
    Average 91 stars, based on 1 article reviews
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    anti mouse factor h  (Hycult Biotech)


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    Hycult Biotech anti mouse factor h
    ( a ) ChPs were stained for C1q (red) and C4 (green). Bar 100 µm. ( b ) C5 siRNA treatment blocks C5 protein deposition in ApoE-/- ChPs; ( c ) ChPs were stained for C3. Ig represents lipid; ( d ) Serum C3 and C5. Serum C3 and C5 protein levels were measured by ELISA. ApoE-/-(n = 6 mice), HFD ApoE4 (n=5). ( e ) High resolution confocal microscopy shows colocalization of ApoE4 (ApoE, red) and Ig (green, represents lipid) in HFD ApoE4-KI ChPs. ApoE-/- ChPs serve as negative controls for ApoE staining; ( f) Complement regulators are expressed in ChPs. WT (n = 5 mice); ApoE-/-(n=4); ND ApoE3 (n=6); HFD ApoE3 (n=6); ND ApoE4 (n=6); HFD ApoE4 (n=6). ( g ) ChP <t>Factor</t> <t>H</t> expressed between WT and ApoE-/- mice. WT (n=5); ApoE-/-(n=4); (h) ChP factor H protein in ChPs. White arrows indicate lipid positive areas. Data in a,b,c,e,h are representative images from at least 3 biologically independent mouse samples. Data in d,f,g represent means ± SEM; Two-tailed Student's t-test was applied to d,g; one-way ANOVA with Tukey posttest was applied to f; Gene names in .
    Anti Mouse Factor H, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 1 article reviews
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    Images

    1) Product Images from "ApoE attenuates unresolvable inflammation by complex formation with activated C1q"

    Article Title: ApoE attenuates unresolvable inflammation by complex formation with activated C1q

    Journal: Nature medicine

    doi: 10.1038/s41591-018-0336-8

    ( a ) ChPs were stained for C1q (red) and C4 (green). Bar 100 µm. ( b ) C5 siRNA treatment blocks C5 protein deposition in ApoE-/- ChPs; ( c ) ChPs were stained for C3. Ig represents lipid; ( d ) Serum C3 and C5. Serum C3 and C5 protein levels were measured by ELISA. ApoE-/-(n = 6 mice), HFD ApoE4 (n=5). ( e ) High resolution confocal microscopy shows colocalization of ApoE4 (ApoE, red) and Ig (green, represents lipid) in HFD ApoE4-KI ChPs. ApoE-/- ChPs serve as negative controls for ApoE staining; ( f) Complement regulators are expressed in ChPs. WT (n = 5 mice); ApoE-/-(n=4); ND ApoE3 (n=6); HFD ApoE3 (n=6); ND ApoE4 (n=6); HFD ApoE4 (n=6). ( g ) ChP Factor H expressed between WT and ApoE-/- mice. WT (n=5); ApoE-/-(n=4); (h) ChP factor H protein in ChPs. White arrows indicate lipid positive areas. Data in a,b,c,e,h are representative images from at least 3 biologically independent mouse samples. Data in d,f,g represent means ± SEM; Two-tailed Student's t-test was applied to d,g; one-way ANOVA with Tukey posttest was applied to f; Gene names in .
    Figure Legend Snippet: ( a ) ChPs were stained for C1q (red) and C4 (green). Bar 100 µm. ( b ) C5 siRNA treatment blocks C5 protein deposition in ApoE-/- ChPs; ( c ) ChPs were stained for C3. Ig represents lipid; ( d ) Serum C3 and C5. Serum C3 and C5 protein levels were measured by ELISA. ApoE-/-(n = 6 mice), HFD ApoE4 (n=5). ( e ) High resolution confocal microscopy shows colocalization of ApoE4 (ApoE, red) and Ig (green, represents lipid) in HFD ApoE4-KI ChPs. ApoE-/- ChPs serve as negative controls for ApoE staining; ( f) Complement regulators are expressed in ChPs. WT (n = 5 mice); ApoE-/-(n=4); ND ApoE3 (n=6); HFD ApoE3 (n=6); ND ApoE4 (n=6); HFD ApoE4 (n=6). ( g ) ChP Factor H expressed between WT and ApoE-/- mice. WT (n=5); ApoE-/-(n=4); (h) ChP factor H protein in ChPs. White arrows indicate lipid positive areas. Data in a,b,c,e,h are representative images from at least 3 biologically independent mouse samples. Data in d,f,g represent means ± SEM; Two-tailed Student's t-test was applied to d,g; one-way ANOVA with Tukey posttest was applied to f; Gene names in .

    Techniques Used: Staining, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Two Tailed Test

    ( a ) ApoE isoforms bind to the C1 complex, but not to C4 or C2. Biotinylated ApoE was immobilized on streptavidin-coated sensors and incubated with C1 complex, C4, C2, or buffer; ( b ) The C1 complex binds to immobilized ApoE isoforms. ( c ) ApoE isoforms bind to C1 and factor H, but not to C3 or C3b; ( d ) Normal human serum (NHS)-derived C1 binds to immobilized plasma-purified ApoE3 and to recombinant ApoE isoforms; ( e ) C1q binds to immobilized plasma-purified ApoE3 and to all recombinant ApoE isoforms; ( f ) Plasma-purified C1q was coated on a sensor chip (CM5) and plasma-derived ApoE (62-1000 nM) was injected into the fluid phase (75 mM NaCl, 5 mM HEPES, 1 mM CaCl2). ( g ) Mannose-binding lectin (MBL) does not bind to C1q as determined by biolayer interferometry; ( h ) Apolipoprotein A (ApoA) does not bind to C1q as determined by biolayer interferometry. ( i ) C1q-ApoE complexes revealed by proximity ligation assay (PLA) on cultured human apoptotic cells (THP-1) were detectable when treated with NHS but not with C1q-depleted serum (dNHS). Data represent mean fluorescence intensity (MFI) ± SEM of 16 cells for each group. Bar 10 µm. Data in b,c,d,e represent means ± SEM of at least three independent experiments. Data a,f,g and h represent means of at least two independent experiments. Two-tailed Student's t-test.
    Figure Legend Snippet: ( a ) ApoE isoforms bind to the C1 complex, but not to C4 or C2. Biotinylated ApoE was immobilized on streptavidin-coated sensors and incubated with C1 complex, C4, C2, or buffer; ( b ) The C1 complex binds to immobilized ApoE isoforms. ( c ) ApoE isoforms bind to C1 and factor H, but not to C3 or C3b; ( d ) Normal human serum (NHS)-derived C1 binds to immobilized plasma-purified ApoE3 and to recombinant ApoE isoforms; ( e ) C1q binds to immobilized plasma-purified ApoE3 and to all recombinant ApoE isoforms; ( f ) Plasma-purified C1q was coated on a sensor chip (CM5) and plasma-derived ApoE (62-1000 nM) was injected into the fluid phase (75 mM NaCl, 5 mM HEPES, 1 mM CaCl2). ( g ) Mannose-binding lectin (MBL) does not bind to C1q as determined by biolayer interferometry; ( h ) Apolipoprotein A (ApoA) does not bind to C1q as determined by biolayer interferometry. ( i ) C1q-ApoE complexes revealed by proximity ligation assay (PLA) on cultured human apoptotic cells (THP-1) were detectable when treated with NHS but not with C1q-depleted serum (dNHS). Data represent mean fluorescence intensity (MFI) ± SEM of 16 cells for each group. Bar 10 µm. Data in b,c,d,e represent means ± SEM of at least three independent experiments. Data a,f,g and h represent means of at least two independent experiments. Two-tailed Student's t-test.

    Techniques Used: Incubation, Derivative Assay, Purification, Recombinant, Injection, Binding Assay, Proximity Ligation Assay, Cell Culture, Fluorescence, Two Tailed Test

    ( a - b ) Human ChP sections were stained for C1q (green) / C3 (red) ( a ) and C1q (green) / ApoE (red) ( b ); ( c - d) ChP sections were stained for CD68+ macrophages/DCs ( c ) and collagen IV (Col-IV) to mark basement membranes. Phase contrast shows lipid deposits in ChPs; ( e ) ChP sections were stained for ApoE (green) and factor H (red); no primary antibody as control (NA). ( f - g ) human brain sections were stained for Aβ (green) / ApoE (red) (left panels), Tau phosphorylation (pTau, green) / ApoE (red) (middle panels), and C1q (green) / ApoE (red) (right panels) (f). Blue for nuclei. No primary antibody as control ( g ); ( h ) brain parenchyma sections were stained for C3 (red) / ApoE (green). Bar 100 µm for a-h; Data in a-h are representative images from at least 3 biologically independent samples.
    Figure Legend Snippet: ( a - b ) Human ChP sections were stained for C1q (green) / C3 (red) ( a ) and C1q (green) / ApoE (red) ( b ); ( c - d) ChP sections were stained for CD68+ macrophages/DCs ( c ) and collagen IV (Col-IV) to mark basement membranes. Phase contrast shows lipid deposits in ChPs; ( e ) ChP sections were stained for ApoE (green) and factor H (red); no primary antibody as control (NA). ( f - g ) human brain sections were stained for Aβ (green) / ApoE (red) (left panels), Tau phosphorylation (pTau, green) / ApoE (red) (middle panels), and C1q (green) / ApoE (red) (right panels) (f). Blue for nuclei. No primary antibody as control ( g ); ( h ) brain parenchyma sections were stained for C3 (red) / ApoE (green). Bar 100 µm for a-h; Data in a-h are representative images from at least 3 biologically independent samples.

    Techniques Used: Staining

    ( a ) Expression microarray analyses of aortas. Heatmaps
show GO terms leukocyte migration, complement activation, phagocytosis, and
cellular response to lipid. 6 weeks WT (n=3 mice); 32 weeks WT (n=3); 6
weeks ApoE-/- (n=3); 32 weeks ApoE-/- (n=3); ( b ) aorta
alternative complement pathway genes (factor B, factor H, factor D) mRNA
expression in 6 weeks and 32 weeks old WT and ApoE-/- mouse aortas. 6 weeks
WT (n=3 mice); 32 weeks WT (n=3); 6 weeks ApoE-/- (n=3); 32 weeks ApoE-/-
(n=3); ( c - d ) plasma cholesterol and body weight;
( e - f ) blood leukocytes and percentage. For
c-f, control (11 mice); C5 siRNA (12 mice). ( g ) blood CD4+ T
cells, CD8+ T cells, and B220+ B cells by flow cytometry. Control (6 mice);
C5 siRNA (6 mice). ( h ) super-resolution microscopy shows
colocalization of C1q (green) and ApoE (red) in human atherosclerotic
plaque. Representative images from at least 3 biologically independent mouse
samples. Bar 5 µm. Data in b,c,d,e,f,g represent means ± SEM;
Two-tailed Student's t-test was applied to c.d.e.f.g; one-way ANOVA with
Tukey posttest was applied to b; abbreviations: WBC, white blood cells; RBC,
red blood cells; PLT, platelets; LYM, lymphocytes; MO, monocytes; GRA,
granulocytes. Gene names and statistics in  .
    Figure Legend Snippet: ( a ) Expression microarray analyses of aortas. Heatmaps show GO terms leukocyte migration, complement activation, phagocytosis, and cellular response to lipid. 6 weeks WT (n=3 mice); 32 weeks WT (n=3); 6 weeks ApoE-/- (n=3); 32 weeks ApoE-/- (n=3); ( b ) aorta alternative complement pathway genes (factor B, factor H, factor D) mRNA expression in 6 weeks and 32 weeks old WT and ApoE-/- mouse aortas. 6 weeks WT (n=3 mice); 32 weeks WT (n=3); 6 weeks ApoE-/- (n=3); 32 weeks ApoE-/- (n=3); ( c - d ) plasma cholesterol and body weight; ( e - f ) blood leukocytes and percentage. For c-f, control (11 mice); C5 siRNA (12 mice). ( g ) blood CD4+ T cells, CD8+ T cells, and B220+ B cells by flow cytometry. Control (6 mice); C5 siRNA (6 mice). ( h ) super-resolution microscopy shows colocalization of C1q (green) and ApoE (red) in human atherosclerotic plaque. Representative images from at least 3 biologically independent mouse samples. Bar 5 µm. Data in b,c,d,e,f,g represent means ± SEM; Two-tailed Student's t-test was applied to c.d.e.f.g; one-way ANOVA with Tukey posttest was applied to b; abbreviations: WBC, white blood cells; RBC, red blood cells; PLT, platelets; LYM, lymphocytes; MO, monocytes; GRA, granulocytes. Gene names and statistics in .

    Techniques Used: Expressing, Microarray, Migration, Activation Assay, Flow Cytometry, Microscopy, Two Tailed Test

    factor h, mouse, mab 1a2, fitc  (Hycult Biotech)


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    Hycult Biotech factor h, mouse, mab 1a2, fitc
    Factor H, Mouse, Mab 1a2, Fitc, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse factor h  (Hycult Biotech)


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    Hycult Biotech mouse factor h
    ( A ) The depicted constructs were used to generate transgenic animals that expressed human C4BP and/or FH in addition to their endogenous counterpart C4BP and FH molecules. ( B ) PCR analysis confirmed the presence of the either hu-C4BP α-chains (upper panel), human <t>Factor</t> <t>H</t> (FH) (lower panel) or both (both panels) in the tg animals, but not in BALB/c wt mice. (C) SDS-PAGE and western blot analysis confirmed that hu-C4BP ( C , upper panel non-reduced gel) and hu-FH ( C , lower panel, reduced gel) were detectable in the appropriately designated tg, but not in BALB/c mouse serum. ( D ) Serum levels of hu-C4BP and hu-FH were determined using a sandwich-ELISA. ( E) BALB/c (blue) and hu-C4BPxFH tg (red) serum (20%) deposit similar amounts of C3 on zymosan particles by flow cytometry. EDTA-treated BALB/c serum (negative control) did not deposit any C3 on zymosan (negative control; n = 3 sera from individual animals). Statistical analysis: Kruskal-Wallis analysis with Dunn’s post-test ( D ) and 1-way ANOVA with Bonferroni’s post-test ( E ).
    Mouse Factor H, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Virulence of Group A Streptococci Is Enhanced by Human Complement Inhibitors"

    Article Title: Virulence of Group A Streptococci Is Enhanced by Human Complement Inhibitors

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005043

    ( A ) The depicted constructs were used to generate transgenic animals that expressed human C4BP and/or FH in addition to their endogenous counterpart C4BP and FH molecules. ( B ) PCR analysis confirmed the presence of the either hu-C4BP α-chains (upper panel), human Factor H (FH) (lower panel) or both (both panels) in the tg animals, but not in BALB/c wt mice. (C) SDS-PAGE and western blot analysis confirmed that hu-C4BP ( C , upper panel non-reduced gel) and hu-FH ( C , lower panel, reduced gel) were detectable in the appropriately designated tg, but not in BALB/c mouse serum. ( D ) Serum levels of hu-C4BP and hu-FH were determined using a sandwich-ELISA. ( E) BALB/c (blue) and hu-C4BPxFH tg (red) serum (20%) deposit similar amounts of C3 on zymosan particles by flow cytometry. EDTA-treated BALB/c serum (negative control) did not deposit any C3 on zymosan (negative control; n = 3 sera from individual animals). Statistical analysis: Kruskal-Wallis analysis with Dunn’s post-test ( D ) and 1-way ANOVA with Bonferroni’s post-test ( E ).
    Figure Legend Snippet: ( A ) The depicted constructs were used to generate transgenic animals that expressed human C4BP and/or FH in addition to their endogenous counterpart C4BP and FH molecules. ( B ) PCR analysis confirmed the presence of the either hu-C4BP α-chains (upper panel), human Factor H (FH) (lower panel) or both (both panels) in the tg animals, but not in BALB/c wt mice. (C) SDS-PAGE and western blot analysis confirmed that hu-C4BP ( C , upper panel non-reduced gel) and hu-FH ( C , lower panel, reduced gel) were detectable in the appropriately designated tg, but not in BALB/c mouse serum. ( D ) Serum levels of hu-C4BP and hu-FH were determined using a sandwich-ELISA. ( E) BALB/c (blue) and hu-C4BPxFH tg (red) serum (20%) deposit similar amounts of C3 on zymosan particles by flow cytometry. EDTA-treated BALB/c serum (negative control) did not deposit any C3 on zymosan (negative control; n = 3 sera from individual animals). Statistical analysis: Kruskal-Wallis analysis with Dunn’s post-test ( D ) and 1-way ANOVA with Bonferroni’s post-test ( E ).

    Techniques Used: Construct, Transgenic Assay, SDS Page, Western Blot, Sandwich ELISA, Flow Cytometry, Negative Control

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    Hycult Biotech complement factor h mouse hycult biotech
    Complement Factor H Mouse Hycult Biotech, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/complement factor h mouse hycult biotech/product/Hycult Biotech
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    complement factor h mouse hycult biotech - by Bioz Stars, 2024-07
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    90
    Hycult Biotech anti mouse factor h
    ( a ) ChPs were stained for C1q (red) and C4 (green). Bar 100 µm. ( b ) C5 siRNA treatment blocks C5 protein deposition in ApoE-/- ChPs; ( c ) ChPs were stained for C3. Ig represents lipid; ( d ) Serum C3 and C5. Serum C3 and C5 protein levels were measured by ELISA. ApoE-/-(n = 6 mice), HFD ApoE4 (n=5). ( e ) High resolution confocal microscopy shows colocalization of ApoE4 (ApoE, red) and Ig (green, represents lipid) in HFD ApoE4-KI ChPs. ApoE-/- ChPs serve as negative controls for ApoE staining; ( f) Complement regulators are expressed in ChPs. WT (n = 5 mice); ApoE-/-(n=4); ND ApoE3 (n=6); HFD ApoE3 (n=6); ND ApoE4 (n=6); HFD ApoE4 (n=6). ( g ) ChP <t>Factor</t> <t>H</t> expressed between WT and ApoE-/- mice. WT (n=5); ApoE-/-(n=4); (h) ChP factor H protein in ChPs. White arrows indicate lipid positive areas. Data in a,b,c,e,h are representative images from at least 3 biologically independent mouse samples. Data in d,f,g represent means ± SEM; Two-tailed Student's t-test was applied to d,g; one-way ANOVA with Tukey posttest was applied to f; Gene names in .
    Anti Mouse Factor H, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti mouse factor h/product/Hycult Biotech
    Average 90 stars, based on 1 article reviews
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    anti mouse factor h - by Bioz Stars, 2024-07
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    90
    Hycult Biotech factor h, mouse, mab 1a2, fitc
    ( a ) ChPs were stained for C1q (red) and C4 (green). Bar 100 µm. ( b ) C5 siRNA treatment blocks C5 protein deposition in ApoE-/- ChPs; ( c ) ChPs were stained for C3. Ig represents lipid; ( d ) Serum C3 and C5. Serum C3 and C5 protein levels were measured by ELISA. ApoE-/-(n = 6 mice), HFD ApoE4 (n=5). ( e ) High resolution confocal microscopy shows colocalization of ApoE4 (ApoE, red) and Ig (green, represents lipid) in HFD ApoE4-KI ChPs. ApoE-/- ChPs serve as negative controls for ApoE staining; ( f) Complement regulators are expressed in ChPs. WT (n = 5 mice); ApoE-/-(n=4); ND ApoE3 (n=6); HFD ApoE3 (n=6); ND ApoE4 (n=6); HFD ApoE4 (n=6). ( g ) ChP <t>Factor</t> <t>H</t> expressed between WT and ApoE-/- mice. WT (n=5); ApoE-/-(n=4); (h) ChP factor H protein in ChPs. White arrows indicate lipid positive areas. Data in a,b,c,e,h are representative images from at least 3 biologically independent mouse samples. Data in d,f,g represent means ± SEM; Two-tailed Student's t-test was applied to d,g; one-way ANOVA with Tukey posttest was applied to f; Gene names in .
    Factor H, Mouse, Mab 1a2, Fitc, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Hycult Biotech mouse factor h
    ( A ) The depicted constructs were used to generate transgenic animals that expressed human C4BP and/or FH in addition to their endogenous counterpart C4BP and FH molecules. ( B ) PCR analysis confirmed the presence of the either hu-C4BP α-chains (upper panel), human <t>Factor</t> <t>H</t> (FH) (lower panel) or both (both panels) in the tg animals, but not in BALB/c wt mice. (C) SDS-PAGE and western blot analysis confirmed that hu-C4BP ( C , upper panel non-reduced gel) and hu-FH ( C , lower panel, reduced gel) were detectable in the appropriately designated tg, but not in BALB/c mouse serum. ( D ) Serum levels of hu-C4BP and hu-FH were determined using a sandwich-ELISA. ( E) BALB/c (blue) and hu-C4BPxFH tg (red) serum (20%) deposit similar amounts of C3 on zymosan particles by flow cytometry. EDTA-treated BALB/c serum (negative control) did not deposit any C3 on zymosan (negative control; n = 3 sera from individual animals). Statistical analysis: Kruskal-Wallis analysis with Dunn’s post-test ( D ) and 1-way ANOVA with Bonferroni’s post-test ( E ).
    Mouse Factor H, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ( a ) ChPs were stained for C1q (red) and C4 (green). Bar 100 µm. ( b ) C5 siRNA treatment blocks C5 protein deposition in ApoE-/- ChPs; ( c ) ChPs were stained for C3. Ig represents lipid; ( d ) Serum C3 and C5. Serum C3 and C5 protein levels were measured by ELISA. ApoE-/-(n = 6 mice), HFD ApoE4 (n=5). ( e ) High resolution confocal microscopy shows colocalization of ApoE4 (ApoE, red) and Ig (green, represents lipid) in HFD ApoE4-KI ChPs. ApoE-/- ChPs serve as negative controls for ApoE staining; ( f) Complement regulators are expressed in ChPs. WT (n = 5 mice); ApoE-/-(n=4); ND ApoE3 (n=6); HFD ApoE3 (n=6); ND ApoE4 (n=6); HFD ApoE4 (n=6). ( g ) ChP Factor H expressed between WT and ApoE-/- mice. WT (n=5); ApoE-/-(n=4); (h) ChP factor H protein in ChPs. White arrows indicate lipid positive areas. Data in a,b,c,e,h are representative images from at least 3 biologically independent mouse samples. Data in d,f,g represent means ± SEM; Two-tailed Student's t-test was applied to d,g; one-way ANOVA with Tukey posttest was applied to f; Gene names in .

    Journal: Nature medicine

    Article Title: ApoE attenuates unresolvable inflammation by complex formation with activated C1q

    doi: 10.1038/s41591-018-0336-8

    Figure Lengend Snippet: ( a ) ChPs were stained for C1q (red) and C4 (green). Bar 100 µm. ( b ) C5 siRNA treatment blocks C5 protein deposition in ApoE-/- ChPs; ( c ) ChPs were stained for C3. Ig represents lipid; ( d ) Serum C3 and C5. Serum C3 and C5 protein levels were measured by ELISA. ApoE-/-(n = 6 mice), HFD ApoE4 (n=5). ( e ) High resolution confocal microscopy shows colocalization of ApoE4 (ApoE, red) and Ig (green, represents lipid) in HFD ApoE4-KI ChPs. ApoE-/- ChPs serve as negative controls for ApoE staining; ( f) Complement regulators are expressed in ChPs. WT (n = 5 mice); ApoE-/-(n=4); ND ApoE3 (n=6); HFD ApoE3 (n=6); ND ApoE4 (n=6); HFD ApoE4 (n=6). ( g ) ChP Factor H expressed between WT and ApoE-/- mice. WT (n=5); ApoE-/-(n=4); (h) ChP factor H protein in ChPs. White arrows indicate lipid positive areas. Data in a,b,c,e,h are representative images from at least 3 biologically independent mouse samples. Data in d,f,g represent means ± SEM; Two-tailed Student's t-test was applied to d,g; one-way ANOVA with Tukey posttest was applied to f; Gene names in .

    Article Snippet: Immunofluorescence staining was performed as previously described , using marker antibodies anti-mouse collagen IV (2150-1470; AbDSerotec), immunoglobulins (715-166-151; Dianova), immunoglobulin isotype that do not react with mouse Ig (017-160-006; Dianova), anti-human/mouse C3 (A213, ComplementTech), anti-human/mouse C3a (A218, ComplementTech), anti-human C5 (A220, ComplementTech), anti-mouse C5 (ab11898, Abcam), isotype for C5 (ab27478, Abcam), anti-mouse C1q (HM1096BT, Hycult Biotech), anti-human C1q (ab71089, Abcam), anti-mouse C4 (HM1046, Hycult Biotech), anti-human Factor H (A312, Quidel), anti-mouse Factor H (HM1119F, Hycult Biotech), anti-human ApoE (ab52607, Abcam), anti-human ApoE (178479; Calbiochem), anti-mouse ApoE (ab183597, Abcam), anti-mouse CD68 (FA11; Serotec), anti-human CD68 (EMB11, DAKO), anti-mouse CD31 (553370; BD PharMingen), anti-mouse iba-1 (019-19741; WAKO), anti-mouse CD45 (BZL 01145; Biozol), anti-human/mouse cytokeratin (Z0622; DAKO), anti-human collagen IV (CIV22, DAKO), anti-beta-amyloid (4G8, Biolegend), anti-phospho-Tau (AT8, Thermofisher), anti-iba1 (polyclonal, WAKO), anti-LAMP1 (1D4B, Abcam), TO-PRO™-3 Iodide (642/661) (T3605, Thermo Fisher Scientific) or DAPI for DNA.

    Techniques: Staining, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Two Tailed Test

    ( a ) ApoE isoforms bind to the C1 complex, but not to C4 or C2. Biotinylated ApoE was immobilized on streptavidin-coated sensors and incubated with C1 complex, C4, C2, or buffer; ( b ) The C1 complex binds to immobilized ApoE isoforms. ( c ) ApoE isoforms bind to C1 and factor H, but not to C3 or C3b; ( d ) Normal human serum (NHS)-derived C1 binds to immobilized plasma-purified ApoE3 and to recombinant ApoE isoforms; ( e ) C1q binds to immobilized plasma-purified ApoE3 and to all recombinant ApoE isoforms; ( f ) Plasma-purified C1q was coated on a sensor chip (CM5) and plasma-derived ApoE (62-1000 nM) was injected into the fluid phase (75 mM NaCl, 5 mM HEPES, 1 mM CaCl2). ( g ) Mannose-binding lectin (MBL) does not bind to C1q as determined by biolayer interferometry; ( h ) Apolipoprotein A (ApoA) does not bind to C1q as determined by biolayer interferometry. ( i ) C1q-ApoE complexes revealed by proximity ligation assay (PLA) on cultured human apoptotic cells (THP-1) were detectable when treated with NHS but not with C1q-depleted serum (dNHS). Data represent mean fluorescence intensity (MFI) ± SEM of 16 cells for each group. Bar 10 µm. Data in b,c,d,e represent means ± SEM of at least three independent experiments. Data a,f,g and h represent means of at least two independent experiments. Two-tailed Student's t-test.

    Journal: Nature medicine

    Article Title: ApoE attenuates unresolvable inflammation by complex formation with activated C1q

    doi: 10.1038/s41591-018-0336-8

    Figure Lengend Snippet: ( a ) ApoE isoforms bind to the C1 complex, but not to C4 or C2. Biotinylated ApoE was immobilized on streptavidin-coated sensors and incubated with C1 complex, C4, C2, or buffer; ( b ) The C1 complex binds to immobilized ApoE isoforms. ( c ) ApoE isoforms bind to C1 and factor H, but not to C3 or C3b; ( d ) Normal human serum (NHS)-derived C1 binds to immobilized plasma-purified ApoE3 and to recombinant ApoE isoforms; ( e ) C1q binds to immobilized plasma-purified ApoE3 and to all recombinant ApoE isoforms; ( f ) Plasma-purified C1q was coated on a sensor chip (CM5) and plasma-derived ApoE (62-1000 nM) was injected into the fluid phase (75 mM NaCl, 5 mM HEPES, 1 mM CaCl2). ( g ) Mannose-binding lectin (MBL) does not bind to C1q as determined by biolayer interferometry; ( h ) Apolipoprotein A (ApoA) does not bind to C1q as determined by biolayer interferometry. ( i ) C1q-ApoE complexes revealed by proximity ligation assay (PLA) on cultured human apoptotic cells (THP-1) were detectable when treated with NHS but not with C1q-depleted serum (dNHS). Data represent mean fluorescence intensity (MFI) ± SEM of 16 cells for each group. Bar 10 µm. Data in b,c,d,e represent means ± SEM of at least three independent experiments. Data a,f,g and h represent means of at least two independent experiments. Two-tailed Student's t-test.

    Article Snippet: Immunofluorescence staining was performed as previously described , using marker antibodies anti-mouse collagen IV (2150-1470; AbDSerotec), immunoglobulins (715-166-151; Dianova), immunoglobulin isotype that do not react with mouse Ig (017-160-006; Dianova), anti-human/mouse C3 (A213, ComplementTech), anti-human/mouse C3a (A218, ComplementTech), anti-human C5 (A220, ComplementTech), anti-mouse C5 (ab11898, Abcam), isotype for C5 (ab27478, Abcam), anti-mouse C1q (HM1096BT, Hycult Biotech), anti-human C1q (ab71089, Abcam), anti-mouse C4 (HM1046, Hycult Biotech), anti-human Factor H (A312, Quidel), anti-mouse Factor H (HM1119F, Hycult Biotech), anti-human ApoE (ab52607, Abcam), anti-human ApoE (178479; Calbiochem), anti-mouse ApoE (ab183597, Abcam), anti-mouse CD68 (FA11; Serotec), anti-human CD68 (EMB11, DAKO), anti-mouse CD31 (553370; BD PharMingen), anti-mouse iba-1 (019-19741; WAKO), anti-mouse CD45 (BZL 01145; Biozol), anti-human/mouse cytokeratin (Z0622; DAKO), anti-human collagen IV (CIV22, DAKO), anti-beta-amyloid (4G8, Biolegend), anti-phospho-Tau (AT8, Thermofisher), anti-iba1 (polyclonal, WAKO), anti-LAMP1 (1D4B, Abcam), TO-PRO™-3 Iodide (642/661) (T3605, Thermo Fisher Scientific) or DAPI for DNA.

    Techniques: Incubation, Derivative Assay, Purification, Recombinant, Injection, Binding Assay, Proximity Ligation Assay, Cell Culture, Fluorescence, Two Tailed Test

    ( a - b ) Human ChP sections were stained for C1q (green) / C3 (red) ( a ) and C1q (green) / ApoE (red) ( b ); ( c - d) ChP sections were stained for CD68+ macrophages/DCs ( c ) and collagen IV (Col-IV) to mark basement membranes. Phase contrast shows lipid deposits in ChPs; ( e ) ChP sections were stained for ApoE (green) and factor H (red); no primary antibody as control (NA). ( f - g ) human brain sections were stained for Aβ (green) / ApoE (red) (left panels), Tau phosphorylation (pTau, green) / ApoE (red) (middle panels), and C1q (green) / ApoE (red) (right panels) (f). Blue for nuclei. No primary antibody as control ( g ); ( h ) brain parenchyma sections were stained for C3 (red) / ApoE (green). Bar 100 µm for a-h; Data in a-h are representative images from at least 3 biologically independent samples.

    Journal: Nature medicine

    Article Title: ApoE attenuates unresolvable inflammation by complex formation with activated C1q

    doi: 10.1038/s41591-018-0336-8

    Figure Lengend Snippet: ( a - b ) Human ChP sections were stained for C1q (green) / C3 (red) ( a ) and C1q (green) / ApoE (red) ( b ); ( c - d) ChP sections were stained for CD68+ macrophages/DCs ( c ) and collagen IV (Col-IV) to mark basement membranes. Phase contrast shows lipid deposits in ChPs; ( e ) ChP sections were stained for ApoE (green) and factor H (red); no primary antibody as control (NA). ( f - g ) human brain sections were stained for Aβ (green) / ApoE (red) (left panels), Tau phosphorylation (pTau, green) / ApoE (red) (middle panels), and C1q (green) / ApoE (red) (right panels) (f). Blue for nuclei. No primary antibody as control ( g ); ( h ) brain parenchyma sections were stained for C3 (red) / ApoE (green). Bar 100 µm for a-h; Data in a-h are representative images from at least 3 biologically independent samples.

    Article Snippet: Immunofluorescence staining was performed as previously described , using marker antibodies anti-mouse collagen IV (2150-1470; AbDSerotec), immunoglobulins (715-166-151; Dianova), immunoglobulin isotype that do not react with mouse Ig (017-160-006; Dianova), anti-human/mouse C3 (A213, ComplementTech), anti-human/mouse C3a (A218, ComplementTech), anti-human C5 (A220, ComplementTech), anti-mouse C5 (ab11898, Abcam), isotype for C5 (ab27478, Abcam), anti-mouse C1q (HM1096BT, Hycult Biotech), anti-human C1q (ab71089, Abcam), anti-mouse C4 (HM1046, Hycult Biotech), anti-human Factor H (A312, Quidel), anti-mouse Factor H (HM1119F, Hycult Biotech), anti-human ApoE (ab52607, Abcam), anti-human ApoE (178479; Calbiochem), anti-mouse ApoE (ab183597, Abcam), anti-mouse CD68 (FA11; Serotec), anti-human CD68 (EMB11, DAKO), anti-mouse CD31 (553370; BD PharMingen), anti-mouse iba-1 (019-19741; WAKO), anti-mouse CD45 (BZL 01145; Biozol), anti-human/mouse cytokeratin (Z0622; DAKO), anti-human collagen IV (CIV22, DAKO), anti-beta-amyloid (4G8, Biolegend), anti-phospho-Tau (AT8, Thermofisher), anti-iba1 (polyclonal, WAKO), anti-LAMP1 (1D4B, Abcam), TO-PRO™-3 Iodide (642/661) (T3605, Thermo Fisher Scientific) or DAPI for DNA.

    Techniques: Staining

    ( a ) Expression microarray analyses of aortas. Heatmaps
show GO terms leukocyte migration, complement activation, phagocytosis, and
cellular response to lipid. 6 weeks WT (n=3 mice); 32 weeks WT (n=3); 6
weeks ApoE-/- (n=3); 32 weeks ApoE-/- (n=3); ( b ) aorta
alternative complement pathway genes (factor B, factor H, factor D) mRNA
expression in 6 weeks and 32 weeks old WT and ApoE-/- mouse aortas. 6 weeks
WT (n=3 mice); 32 weeks WT (n=3); 6 weeks ApoE-/- (n=3); 32 weeks ApoE-/-
(n=3); ( c - d ) plasma cholesterol and body weight;
( e - f ) blood leukocytes and percentage. For
c-f, control (11 mice); C5 siRNA (12 mice). ( g ) blood CD4+ T
cells, CD8+ T cells, and B220+ B cells by flow cytometry. Control (6 mice);
C5 siRNA (6 mice). ( h ) super-resolution microscopy shows
colocalization of C1q (green) and ApoE (red) in human atherosclerotic
plaque. Representative images from at least 3 biologically independent mouse
samples. Bar 5 µm. Data in b,c,d,e,f,g represent means ± SEM;
Two-tailed Student's t-test was applied to c.d.e.f.g; one-way ANOVA with
Tukey posttest was applied to b; abbreviations: WBC, white blood cells; RBC,
red blood cells; PLT, platelets; LYM, lymphocytes; MO, monocytes; GRA,
granulocytes. Gene names and statistics in  .

    Journal: Nature medicine

    Article Title: ApoE attenuates unresolvable inflammation by complex formation with activated C1q

    doi: 10.1038/s41591-018-0336-8

    Figure Lengend Snippet: ( a ) Expression microarray analyses of aortas. Heatmaps show GO terms leukocyte migration, complement activation, phagocytosis, and cellular response to lipid. 6 weeks WT (n=3 mice); 32 weeks WT (n=3); 6 weeks ApoE-/- (n=3); 32 weeks ApoE-/- (n=3); ( b ) aorta alternative complement pathway genes (factor B, factor H, factor D) mRNA expression in 6 weeks and 32 weeks old WT and ApoE-/- mouse aortas. 6 weeks WT (n=3 mice); 32 weeks WT (n=3); 6 weeks ApoE-/- (n=3); 32 weeks ApoE-/- (n=3); ( c - d ) plasma cholesterol and body weight; ( e - f ) blood leukocytes and percentage. For c-f, control (11 mice); C5 siRNA (12 mice). ( g ) blood CD4+ T cells, CD8+ T cells, and B220+ B cells by flow cytometry. Control (6 mice); C5 siRNA (6 mice). ( h ) super-resolution microscopy shows colocalization of C1q (green) and ApoE (red) in human atherosclerotic plaque. Representative images from at least 3 biologically independent mouse samples. Bar 5 µm. Data in b,c,d,e,f,g represent means ± SEM; Two-tailed Student's t-test was applied to c.d.e.f.g; one-way ANOVA with Tukey posttest was applied to b; abbreviations: WBC, white blood cells; RBC, red blood cells; PLT, platelets; LYM, lymphocytes; MO, monocytes; GRA, granulocytes. Gene names and statistics in .

    Article Snippet: Immunofluorescence staining was performed as previously described , using marker antibodies anti-mouse collagen IV (2150-1470; AbDSerotec), immunoglobulins (715-166-151; Dianova), immunoglobulin isotype that do not react with mouse Ig (017-160-006; Dianova), anti-human/mouse C3 (A213, ComplementTech), anti-human/mouse C3a (A218, ComplementTech), anti-human C5 (A220, ComplementTech), anti-mouse C5 (ab11898, Abcam), isotype for C5 (ab27478, Abcam), anti-mouse C1q (HM1096BT, Hycult Biotech), anti-human C1q (ab71089, Abcam), anti-mouse C4 (HM1046, Hycult Biotech), anti-human Factor H (A312, Quidel), anti-mouse Factor H (HM1119F, Hycult Biotech), anti-human ApoE (ab52607, Abcam), anti-human ApoE (178479; Calbiochem), anti-mouse ApoE (ab183597, Abcam), anti-mouse CD68 (FA11; Serotec), anti-human CD68 (EMB11, DAKO), anti-mouse CD31 (553370; BD PharMingen), anti-mouse iba-1 (019-19741; WAKO), anti-mouse CD45 (BZL 01145; Biozol), anti-human/mouse cytokeratin (Z0622; DAKO), anti-human collagen IV (CIV22, DAKO), anti-beta-amyloid (4G8, Biolegend), anti-phospho-Tau (AT8, Thermofisher), anti-iba1 (polyclonal, WAKO), anti-LAMP1 (1D4B, Abcam), TO-PRO™-3 Iodide (642/661) (T3605, Thermo Fisher Scientific) or DAPI for DNA.

    Techniques: Expressing, Microarray, Migration, Activation Assay, Flow Cytometry, Microscopy, Two Tailed Test

    ( A ) The depicted constructs were used to generate transgenic animals that expressed human C4BP and/or FH in addition to their endogenous counterpart C4BP and FH molecules. ( B ) PCR analysis confirmed the presence of the either hu-C4BP α-chains (upper panel), human Factor H (FH) (lower panel) or both (both panels) in the tg animals, but not in BALB/c wt mice. (C) SDS-PAGE and western blot analysis confirmed that hu-C4BP ( C , upper panel non-reduced gel) and hu-FH ( C , lower panel, reduced gel) were detectable in the appropriately designated tg, but not in BALB/c mouse serum. ( D ) Serum levels of hu-C4BP and hu-FH were determined using a sandwich-ELISA. ( E) BALB/c (blue) and hu-C4BPxFH tg (red) serum (20%) deposit similar amounts of C3 on zymosan particles by flow cytometry. EDTA-treated BALB/c serum (negative control) did not deposit any C3 on zymosan (negative control; n = 3 sera from individual animals). Statistical analysis: Kruskal-Wallis analysis with Dunn’s post-test ( D ) and 1-way ANOVA with Bonferroni’s post-test ( E ).

    Journal: PLoS Pathogens

    Article Title: Virulence of Group A Streptococci Is Enhanced by Human Complement Inhibitors

    doi: 10.1371/journal.ppat.1005043

    Figure Lengend Snippet: ( A ) The depicted constructs were used to generate transgenic animals that expressed human C4BP and/or FH in addition to their endogenous counterpart C4BP and FH molecules. ( B ) PCR analysis confirmed the presence of the either hu-C4BP α-chains (upper panel), human Factor H (FH) (lower panel) or both (both panels) in the tg animals, but not in BALB/c wt mice. (C) SDS-PAGE and western blot analysis confirmed that hu-C4BP ( C , upper panel non-reduced gel) and hu-FH ( C , lower panel, reduced gel) were detectable in the appropriately designated tg, but not in BALB/c mouse serum. ( D ) Serum levels of hu-C4BP and hu-FH were determined using a sandwich-ELISA. ( E) BALB/c (blue) and hu-C4BPxFH tg (red) serum (20%) deposit similar amounts of C3 on zymosan particles by flow cytometry. EDTA-treated BALB/c serum (negative control) did not deposit any C3 on zymosan (negative control; n = 3 sera from individual animals). Statistical analysis: Kruskal-Wallis analysis with Dunn’s post-test ( D ) and 1-way ANOVA with Bonferroni’s post-test ( E ).

    Article Snippet: For flow cytometry analysis, the following antibodies were used: mouse anti human-C4BP MK104 either unconjugated or conjugated to biotin; mouse anti human-Factor H MRC OX24 unconjugated or conjugated to biotin; rabbit anti mouse-C4BP (homemade) conjugated to Dylight 647; mouse monoclonal anti mouse-Factor H (Hycult, HM1119) conjugated to biotin; goat anti mouse-C3c (Nordic Immunology, GAM/C3c/7S); anti mouse C3 FITC (MP Biomedicals #0855500) anti mouse Ly-6G brilliant violet 421 (BioLegend, #127627); anti mouse Ly-6C PerCP/Cy5.5 (BioLegend, #128011); anti mouse CD11c (BioLegend, #117317); anti mouse I-A/I-E brilliant violet 510 (BioLegend, #107635); anti mouse CD64 APC (BioLegend, #139305); anti mouse/human CD11b APC/Cy7 (BioLegend, #101225).

    Techniques: Construct, Transgenic Assay, SDS Page, Western Blot, Sandwich ELISA, Flow Cytometry, Negative Control