Structured Review

Abcam filipin
Npc1 -/- mice liver and NPC cells show increased MLN64 expression. (A) To detect protein expression 30 μg of protein from liver homogenates were subjected to SDS-PAGE and western blotting for MLN64. ε-COP was used as a loading control. The Figure shows a western blot representative image, n=5. (B) Western blots bands intensity quantification. (C) Real time PCR analysis of MLN64 mRNA from mouse liver, n=4. (D) 30 μg of protein of CHO-WT and CHO-NPC cell extracts were analyzed by western blot against MLN64 and ε-COP. A representative image is shown, n=3. (E) Western blots bands intensity quantification. (F) MLN64 immunofluorescence analysis (Top). CHO-WT and CHO-NPC cells were immunostained with an anti-MLN64 antibody. <t>Filipin</t> staining (Bottom). CHO-WT and CHO-NPC cells were fixed, and cholesterol accumulation was detected by filipin staining. Scale bar: 20 µm (G) Graph shows quantifications of fluorescence. * Indicates statistically significant differences (p
Filipin, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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93/100 stars

Images

1) Product Images from "MLN64 induces mitochondrial dysfunction associated with increased mitochondrial cholesterol content"

Article Title: MLN64 induces mitochondrial dysfunction associated with increased mitochondrial cholesterol content

Journal: Redox Biology

doi: 10.1016/j.redox.2017.02.024

Npc1 -/- mice liver and NPC cells show increased MLN64 expression. (A) To detect protein expression 30 μg of protein from liver homogenates were subjected to SDS-PAGE and western blotting for MLN64. ε-COP was used as a loading control. The Figure shows a western blot representative image, n=5. (B) Western blots bands intensity quantification. (C) Real time PCR analysis of MLN64 mRNA from mouse liver, n=4. (D) 30 μg of protein of CHO-WT and CHO-NPC cell extracts were analyzed by western blot against MLN64 and ε-COP. A representative image is shown, n=3. (E) Western blots bands intensity quantification. (F) MLN64 immunofluorescence analysis (Top). CHO-WT and CHO-NPC cells were immunostained with an anti-MLN64 antibody. Filipin staining (Bottom). CHO-WT and CHO-NPC cells were fixed, and cholesterol accumulation was detected by filipin staining. Scale bar: 20 µm (G) Graph shows quantifications of fluorescence. * Indicates statistically significant differences (p
Figure Legend Snippet: Npc1 -/- mice liver and NPC cells show increased MLN64 expression. (A) To detect protein expression 30 μg of protein from liver homogenates were subjected to SDS-PAGE and western blotting for MLN64. ε-COP was used as a loading control. The Figure shows a western blot representative image, n=5. (B) Western blots bands intensity quantification. (C) Real time PCR analysis of MLN64 mRNA from mouse liver, n=4. (D) 30 μg of protein of CHO-WT and CHO-NPC cell extracts were analyzed by western blot against MLN64 and ε-COP. A representative image is shown, n=3. (E) Western blots bands intensity quantification. (F) MLN64 immunofluorescence analysis (Top). CHO-WT and CHO-NPC cells were immunostained with an anti-MLN64 antibody. Filipin staining (Bottom). CHO-WT and CHO-NPC cells were fixed, and cholesterol accumulation was detected by filipin staining. Scale bar: 20 µm (G) Graph shows quantifications of fluorescence. * Indicates statistically significant differences (p

Techniques Used: Mouse Assay, Expressing, SDS Page, Western Blot, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Fluorescence

MLN64 overexpression increases mitochondrial cholesterol and decreases mitochondrial glutathione and ATPase activity in cultured hepatocytes (A) Immunofluorescence and filipin staining analysis of Ad.E1Δ and Ad.MLN64 HepG2 cells stained with anti-MLN64 (MLN64, red), anti-cytochrome c (Cyt-C, green) and filipin (cyan) 48 h after infection. Colocalization of Cyt-C and filipin indicating of mitochondrial cholesterol is depicted (white). Merge and zoom of the merge. Scale bar: 20 µm. (B) The graph shows the Pearson’s coefficient that measures the percentage of colocalization of filipin and Cyt-C. The photographs shown are representative confocal images, (n=3). (C) Mitochondrial glutathione content. Mitochondria were isolated by Percoll density ultracentrifugation from Ad.E1Δ and Ad.MLN64 HepG2 cells 48 h after infection. Glutathione in mitochondria was determined as described in the Materials and Methods section, (n=4). (D) ATPase activity in mitochondrial fraction from HepG2 cells (n=4) was quantified by measuring the hydrolysis rate of ATP. *Indicates statistically significant differences (p
Figure Legend Snippet: MLN64 overexpression increases mitochondrial cholesterol and decreases mitochondrial glutathione and ATPase activity in cultured hepatocytes (A) Immunofluorescence and filipin staining analysis of Ad.E1Δ and Ad.MLN64 HepG2 cells stained with anti-MLN64 (MLN64, red), anti-cytochrome c (Cyt-C, green) and filipin (cyan) 48 h after infection. Colocalization of Cyt-C and filipin indicating of mitochondrial cholesterol is depicted (white). Merge and zoom of the merge. Scale bar: 20 µm. (B) The graph shows the Pearson’s coefficient that measures the percentage of colocalization of filipin and Cyt-C. The photographs shown are representative confocal images, (n=3). (C) Mitochondrial glutathione content. Mitochondria were isolated by Percoll density ultracentrifugation from Ad.E1Δ and Ad.MLN64 HepG2 cells 48 h after infection. Glutathione in mitochondria was determined as described in the Materials and Methods section, (n=4). (D) ATPase activity in mitochondrial fraction from HepG2 cells (n=4) was quantified by measuring the hydrolysis rate of ATP. *Indicates statistically significant differences (p

Techniques Used: Over Expression, Activity Assay, Cell Culture, Immunofluorescence, Staining, Infection, Isolation

2) Product Images from "ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism"

Article Title: ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism

Journal: Brain

doi: 10.1093/brain/awx094

Abnormal cholesterol homeostasis and reduced expression of OXPHOS factors associated with ATAD3 gene cluster deletions. ( A ) Rank gene expression list showing the position of selected factors for Subjects S1a and S5 versus control (see also Supplementary Figs 8–10 and Supplementary material ). ( B and C ) Proteins from fibroblasts of controls and S1a and S5 were fractionated by SDS-PAGE and probed with the indicated antibodies after blot transfer. There was no increase in the steady-state level of the larger isoforms of SREBF2 (located in the cytoplasm, SREBF2-C) among the subject samples but a SCAP processed SREBF2 isoform, which translocates to the nucleus (SREBF2-N) and resolves at ∼55 kDa, was increased in S1a and S5 samples (this particular species may be a phosphorylated form of SREBF2 ( Krycer et al. , 2012 ); CES1 was greatly diminished. ( D and E ) Free cholesterol detected by filipin labelling of fibroblasts exposed to ( D ) no treatment, or ( E ) 5 μM U18666A for 72 h. * P
Figure Legend Snippet: Abnormal cholesterol homeostasis and reduced expression of OXPHOS factors associated with ATAD3 gene cluster deletions. ( A ) Rank gene expression list showing the position of selected factors for Subjects S1a and S5 versus control (see also Supplementary Figs 8–10 and Supplementary material ). ( B and C ) Proteins from fibroblasts of controls and S1a and S5 were fractionated by SDS-PAGE and probed with the indicated antibodies after blot transfer. There was no increase in the steady-state level of the larger isoforms of SREBF2 (located in the cytoplasm, SREBF2-C) among the subject samples but a SCAP processed SREBF2 isoform, which translocates to the nucleus (SREBF2-N) and resolves at ∼55 kDa, was increased in S1a and S5 samples (this particular species may be a phosphorylated form of SREBF2 ( Krycer et al. , 2012 ); CES1 was greatly diminished. ( D and E ) Free cholesterol detected by filipin labelling of fibroblasts exposed to ( D ) no treatment, or ( E ) 5 μM U18666A for 72 h. * P

Techniques Used: Expressing, SDS Page

3) Product Images from "Atypical antipsychotics alter cholesterol and fatty acid metabolism in vitro [S]"

Article Title: Atypical antipsychotics alter cholesterol and fatty acid metabolism in vitro [S]

Journal: Journal of Lipid Research

doi: 10.1194/jlr.M026948

Effects of antipsychotics on endosome/lysosome accumulation of LDL. Untreated HepG2 cells (control) and cells treated for 24 h with haloperidol, clozapine, risperidone, and ziprasidone 10 µM were incubated with DiI-LDL (30 µg/ml of cholesterol). A: At the end of the incubation period, cells were fixed and stained with filipin and LAMP2 and then photographed using a confocal microscope. B: At the end of the incubation period, cells were fixed and stained with filipin and CD63 and then photographed using a confocal microscope. Results from a representative experiment of three independent experiments are shown.
Figure Legend Snippet: Effects of antipsychotics on endosome/lysosome accumulation of LDL. Untreated HepG2 cells (control) and cells treated for 24 h with haloperidol, clozapine, risperidone, and ziprasidone 10 µM were incubated with DiI-LDL (30 µg/ml of cholesterol). A: At the end of the incubation period, cells were fixed and stained with filipin and LAMP2 and then photographed using a confocal microscope. B: At the end of the incubation period, cells were fixed and stained with filipin and CD63 and then photographed using a confocal microscope. Results from a representative experiment of three independent experiments are shown.

Techniques Used: Incubation, Staining, Microscopy

Related Articles

Flow Cytometry:

Article Title: High density lipoproteins selectively promote the survival of human regulatory T cells [S]
Article Snippet: Paragraph title: Flow cytometry ... Free cholesterol content was quantified by staining with filipin III, following the manufacturer’s instructions (cholesterol assay kit; Abcam, Cambridge, MA).

Immunocytochemistry:

Article Title: ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism
Article Snippet: Paragraph title: Immunocytochemistry and cell imaging ... Unesterified cholesterol in fibroblasts was stained with filipin, using a cholesterol assay kit (Abcam), detected by wide-field fluorescence microscopy and quantified using ImageJ.

Fluorescence:

Article Title: ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism
Article Snippet: .. Unesterified cholesterol in fibroblasts was stained with filipin, using a cholesterol assay kit (Abcam), detected by wide-field fluorescence microscopy and quantified using ImageJ. .. Mitochondrial DNA foci sizing and counting Nucleoid size was analysed using 3D confocal images of human fibroblast cells stained with anti-DNA antibody.

Cytometry:

Article Title: High density lipoproteins selectively promote the survival of human regulatory T cells [S]
Article Snippet: Paragraph title: Flow cytometry ... Free cholesterol content was quantified by staining with filipin III, following the manufacturer’s instructions (cholesterol assay kit; Abcam, Cambridge, MA).

Microscopy:

Article Title: MLN64 induces mitochondrial dysfunction associated with increased mitochondrial cholesterol content
Article Snippet: Cells were incubated for 2 h with 25 μg/ml filipin, rinsed with PBS, followed by incubation with primary antibodies, mouse anti-cytochrome C antibody (1:200, ab13575, Abcam) and rabbit anti-MLN64 polyclonal antibody (1:500), rinsed with PBS, and incubated for 2 h with secondary antibodies (1:1000). .. After the final washes in PBS, cells were mounted and confocal images acquired using a Leica SP2 laser scanning confocal microscope.

Article Title: ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism
Article Snippet: .. Unesterified cholesterol in fibroblasts was stained with filipin, using a cholesterol assay kit (Abcam), detected by wide-field fluorescence microscopy and quantified using ImageJ. .. Mitochondrial DNA foci sizing and counting Nucleoid size was analysed using 3D confocal images of human fibroblast cells stained with anti-DNA antibody.

Article Title: Atypical antipsychotics alter cholesterol and fatty acid metabolism in vitro [S]
Article Snippet: Paragraph title: Immunofluorescence microscopy ... HepG2 cells were cultured on glass coverslips and fixed with 4% paraformaldehyde/PBS for 5 min. Next, the cells were permeabilized in 0.1% triton X-100/PBS for 5 min and incubated with 2% BSA (BSA) in PBS for 45 min. For free cholesterol staining, cells were exposed to filipin (50 mg/l in PBS) for 45 min. Anti-Lamp2 (Abcam, Cambridge, UK) or anti-CD63 (BD Biosciences) were used at a 1:200 dilution for 2 h, followed by incubation with Alexa fluor 488-conjugated anti-mouse IgG (Molecular Probes, Invitrogen S.A.) in PBS at a 1:500 dilution for 45 min.

Incubation:

Article Title: MLN64 induces mitochondrial dysfunction associated with increased mitochondrial cholesterol content
Article Snippet: .. Cells were incubated for 2 h with 25 μg/ml filipin, rinsed with PBS, followed by incubation with primary antibodies, mouse anti-cytochrome C antibody (1:200, ab13575, Abcam) and rabbit anti-MLN64 polyclonal antibody (1:500), rinsed with PBS, and incubated for 2 h with secondary antibodies (1:1000). ..

Article Title: ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism
Article Snippet: A 90 min incubation at room temperature with Alexa Fluor® 488 conjugated streptavidin (Invitrogen) was followed with a final set of PBS washes. .. Unesterified cholesterol in fibroblasts was stained with filipin, using a cholesterol assay kit (Abcam), detected by wide-field fluorescence microscopy and quantified using ImageJ.

Article Title: High density lipoproteins selectively promote the survival of human regulatory T cells [S]
Article Snippet: To evaluate the cholesteryl ester (CE) content by flow cytometry, cells were incubated for 15 min at 4°C in the dark with cholesteryl BODIPY® FL C12 (500 ng/ml; Invitrogen, Grand Island, NY). .. Free cholesterol content was quantified by staining with filipin III, following the manufacturer’s instructions (cholesterol assay kit; Abcam, Cambridge, MA).

Article Title: Atypical antipsychotics alter cholesterol and fatty acid metabolism in vitro [S]
Article Snippet: .. HepG2 cells were cultured on glass coverslips and fixed with 4% paraformaldehyde/PBS for 5 min. Next, the cells were permeabilized in 0.1% triton X-100/PBS for 5 min and incubated with 2% BSA (BSA) in PBS for 45 min. For free cholesterol staining, cells were exposed to filipin (50 mg/l in PBS) for 45 min. Anti-Lamp2 (Abcam, Cambridge, UK) or anti-CD63 (BD Biosciences) were used at a 1:200 dilution for 2 h, followed by incubation with Alexa fluor 488-conjugated anti-mouse IgG (Molecular Probes, Invitrogen S.A.) in PBS at a 1:500 dilution for 45 min. .. Cells were mounted for microscopy and examined on a Nikon D Eclipse C1 confocal microscope.

Cell Culture:

Article Title: MLN64 induces mitochondrial dysfunction associated with increased mitochondrial cholesterol content
Article Snippet: 2.7 Laser confocal imaging For immunofluorescence, cultured HepG2 cells were fixed in 4% paraformaldehyde in PBS for 10 min. .. Cells were incubated for 2 h with 25 μg/ml filipin, rinsed with PBS, followed by incubation with primary antibodies, mouse anti-cytochrome C antibody (1:200, ab13575, Abcam) and rabbit anti-MLN64 polyclonal antibody (1:500), rinsed with PBS, and incubated for 2 h with secondary antibodies (1:1000).

Article Title: Atypical antipsychotics alter cholesterol and fatty acid metabolism in vitro [S]
Article Snippet: .. HepG2 cells were cultured on glass coverslips and fixed with 4% paraformaldehyde/PBS for 5 min. Next, the cells were permeabilized in 0.1% triton X-100/PBS for 5 min and incubated with 2% BSA (BSA) in PBS for 45 min. For free cholesterol staining, cells were exposed to filipin (50 mg/l in PBS) for 45 min. Anti-Lamp2 (Abcam, Cambridge, UK) or anti-CD63 (BD Biosciences) were used at a 1:200 dilution for 2 h, followed by incubation with Alexa fluor 488-conjugated anti-mouse IgG (Molecular Probes, Invitrogen S.A.) in PBS at a 1:500 dilution for 45 min. .. Cells were mounted for microscopy and examined on a Nikon D Eclipse C1 confocal microscope.

Labeling:

Article Title: High density lipoproteins selectively promote the survival of human regulatory T cells [S]
Article Snippet: Free cholesterol content was quantified by staining with filipin III, following the manufacturer’s instructions (cholesterol assay kit; Abcam, Cambridge, MA). .. In addition, the rabbit blocking SR-BI/II polyclonal antibody (Novus Biological) was labeled with Zenon AF700 (Life Technology, Grand Island, NY) and used to determine levels of SR-BI/II expression.

Expressing:

Article Title: High density lipoproteins selectively promote the survival of human regulatory T cells [S]
Article Snippet: Free cholesterol content was quantified by staining with filipin III, following the manufacturer’s instructions (cholesterol assay kit; Abcam, Cambridge, MA). .. In addition, the rabbit blocking SR-BI/II polyclonal antibody (Novus Biological) was labeled with Zenon AF700 (Life Technology, Grand Island, NY) and used to determine levels of SR-BI/II expression.

Staining:

Article Title: ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism
Article Snippet: .. Unesterified cholesterol in fibroblasts was stained with filipin, using a cholesterol assay kit (Abcam), detected by wide-field fluorescence microscopy and quantified using ImageJ. .. Mitochondrial DNA foci sizing and counting Nucleoid size was analysed using 3D confocal images of human fibroblast cells stained with anti-DNA antibody.

Article Title: High density lipoproteins selectively promote the survival of human regulatory T cells [S]
Article Snippet: .. Free cholesterol content was quantified by staining with filipin III, following the manufacturer’s instructions (cholesterol assay kit; Abcam, Cambridge, MA). .. In addition, the rabbit blocking SR-BI/II polyclonal antibody (Novus Biological) was labeled with Zenon AF700 (Life Technology, Grand Island, NY) and used to determine levels of SR-BI/II expression.

Article Title: Atypical antipsychotics alter cholesterol and fatty acid metabolism in vitro [S]
Article Snippet: .. HepG2 cells were cultured on glass coverslips and fixed with 4% paraformaldehyde/PBS for 5 min. Next, the cells were permeabilized in 0.1% triton X-100/PBS for 5 min and incubated with 2% BSA (BSA) in PBS for 45 min. For free cholesterol staining, cells were exposed to filipin (50 mg/l in PBS) for 45 min. Anti-Lamp2 (Abcam, Cambridge, UK) or anti-CD63 (BD Biosciences) were used at a 1:200 dilution for 2 h, followed by incubation with Alexa fluor 488-conjugated anti-mouse IgG (Molecular Probes, Invitrogen S.A.) in PBS at a 1:500 dilution for 45 min. .. Cells were mounted for microscopy and examined on a Nikon D Eclipse C1 confocal microscope.

Cholesterol Assay:

Article Title: ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism
Article Snippet: .. Unesterified cholesterol in fibroblasts was stained with filipin, using a cholesterol assay kit (Abcam), detected by wide-field fluorescence microscopy and quantified using ImageJ. .. Mitochondrial DNA foci sizing and counting Nucleoid size was analysed using 3D confocal images of human fibroblast cells stained with anti-DNA antibody.

Article Title: High density lipoproteins selectively promote the survival of human regulatory T cells [S]
Article Snippet: .. Free cholesterol content was quantified by staining with filipin III, following the manufacturer’s instructions (cholesterol assay kit; Abcam, Cambridge, MA). .. In addition, the rabbit blocking SR-BI/II polyclonal antibody (Novus Biological) was labeled with Zenon AF700 (Life Technology, Grand Island, NY) and used to determine levels of SR-BI/II expression.

Immunofluorescence:

Article Title: MLN64 induces mitochondrial dysfunction associated with increased mitochondrial cholesterol content
Article Snippet: 2.7 Laser confocal imaging For immunofluorescence, cultured HepG2 cells were fixed in 4% paraformaldehyde in PBS for 10 min. .. Cells were incubated for 2 h with 25 μg/ml filipin, rinsed with PBS, followed by incubation with primary antibodies, mouse anti-cytochrome C antibody (1:200, ab13575, Abcam) and rabbit anti-MLN64 polyclonal antibody (1:500), rinsed with PBS, and incubated for 2 h with secondary antibodies (1:1000).

Article Title: Atypical antipsychotics alter cholesterol and fatty acid metabolism in vitro [S]
Article Snippet: Paragraph title: Immunofluorescence microscopy ... HepG2 cells were cultured on glass coverslips and fixed with 4% paraformaldehyde/PBS for 5 min. Next, the cells were permeabilized in 0.1% triton X-100/PBS for 5 min and incubated with 2% BSA (BSA) in PBS for 45 min. For free cholesterol staining, cells were exposed to filipin (50 mg/l in PBS) for 45 min. Anti-Lamp2 (Abcam, Cambridge, UK) or anti-CD63 (BD Biosciences) were used at a 1:200 dilution for 2 h, followed by incubation with Alexa fluor 488-conjugated anti-mouse IgG (Molecular Probes, Invitrogen S.A.) in PBS at a 1:500 dilution for 45 min.

Blocking Assay:

Article Title: High density lipoproteins selectively promote the survival of human regulatory T cells [S]
Article Snippet: Free cholesterol content was quantified by staining with filipin III, following the manufacturer’s instructions (cholesterol assay kit; Abcam, Cambridge, MA). .. In addition, the rabbit blocking SR-BI/II polyclonal antibody (Novus Biological) was labeled with Zenon AF700 (Life Technology, Grand Island, NY) and used to determine levels of SR-BI/II expression.

Imaging:

Article Title: MLN64 induces mitochondrial dysfunction associated with increased mitochondrial cholesterol content
Article Snippet: Paragraph title: Laser confocal imaging ... Cells were incubated for 2 h with 25 μg/ml filipin, rinsed with PBS, followed by incubation with primary antibodies, mouse anti-cytochrome C antibody (1:200, ab13575, Abcam) and rabbit anti-MLN64 polyclonal antibody (1:500), rinsed with PBS, and incubated for 2 h with secondary antibodies (1:1000).

Article Title: ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism
Article Snippet: Paragraph title: Immunocytochemistry and cell imaging ... Unesterified cholesterol in fibroblasts was stained with filipin, using a cholesterol assay kit (Abcam), detected by wide-field fluorescence microscopy and quantified using ImageJ.

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    Abcam filipin staining
    Colocalization of human wild-type APP with cholesterol in CHO/APP wt cells after mAb ED-C99 treatment. ( a ) CHO/APP wt cells were plated to 8-well slide chamber and then, after overnight incubation, the cells were treated with mAb ED-C99 or IgG 1 isotype control for 2 h. These cells were stained by <t>Filipin</t> in strict accordance with the manufacturer's instructions of the cholesterol assay kit. ( b ) After Filipin staining and washing, some of these cells were permeabilized with 0.05% Triton X-100 for 5 min, washed, and stained with rabbit anti-APP-C-terminal antibody overnight at 4°C. Alexa Fluor 594 Donkey anti-rabbit IgG was used to detect APP signals. Confocal images were taken by Olympus fluoview FV1000 laser scanning confocal microscope (Tokyo, Japan)
    Filipin Staining, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/filipin staining/product/Abcam
    Average 95 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    filipin staining - by Bioz Stars, 2020-04
    95/100 stars
      Buy from Supplier

    93
    Abcam filipin
    Npc1 -/- mice liver and NPC cells show increased MLN64 expression. (A) To detect protein expression 30 μg of protein from liver homogenates were subjected to SDS-PAGE and western blotting for MLN64. ε-COP was used as a loading control. The Figure shows a western blot representative image, n=5. (B) Western blots bands intensity quantification. (C) Real time PCR analysis of MLN64 mRNA from mouse liver, n=4. (D) 30 μg of protein of CHO-WT and CHO-NPC cell extracts were analyzed by western blot against MLN64 and ε-COP. A representative image is shown, n=3. (E) Western blots bands intensity quantification. (F) MLN64 immunofluorescence analysis (Top). CHO-WT and CHO-NPC cells were immunostained with an anti-MLN64 antibody. <t>Filipin</t> staining (Bottom). CHO-WT and CHO-NPC cells were fixed, and cholesterol accumulation was detected by filipin staining. Scale bar: 20 µm (G) Graph shows quantifications of fluorescence. * Indicates statistically significant differences (p
    Filipin, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/filipin/product/Abcam
    Average 93 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    filipin - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

    Image Search Results


    Colocalization of human wild-type APP with cholesterol in CHO/APP wt cells after mAb ED-C99 treatment. ( a ) CHO/APP wt cells were plated to 8-well slide chamber and then, after overnight incubation, the cells were treated with mAb ED-C99 or IgG 1 isotype control for 2 h. These cells were stained by Filipin in strict accordance with the manufacturer's instructions of the cholesterol assay kit. ( b ) After Filipin staining and washing, some of these cells were permeabilized with 0.05% Triton X-100 for 5 min, washed, and stained with rabbit anti-APP-C-terminal antibody overnight at 4°C. Alexa Fluor 594 Donkey anti-rabbit IgG was used to detect APP signals. Confocal images were taken by Olympus fluoview FV1000 laser scanning confocal microscope (Tokyo, Japan)

    Journal: Cell Death & Disease

    Article Title: Specific antibody binding to the APP672–699 region shifts APP processing from α- to β-cleavage

    doi: 10.1038/cddis.2014.336

    Figure Lengend Snippet: Colocalization of human wild-type APP with cholesterol in CHO/APP wt cells after mAb ED-C99 treatment. ( a ) CHO/APP wt cells were plated to 8-well slide chamber and then, after overnight incubation, the cells were treated with mAb ED-C99 or IgG 1 isotype control for 2 h. These cells were stained by Filipin in strict accordance with the manufacturer's instructions of the cholesterol assay kit. ( b ) After Filipin staining and washing, some of these cells were permeabilized with 0.05% Triton X-100 for 5 min, washed, and stained with rabbit anti-APP-C-terminal antibody overnight at 4°C. Alexa Fluor 594 Donkey anti-rabbit IgG was used to detect APP signals. Confocal images were taken by Olympus fluoview FV1000 laser scanning confocal microscope (Tokyo, Japan)

    Article Snippet: Filipin staining After treatment, the cells were fixed and Filipin staining was performed following the manufacturer's instructions of the cholesterol assay kit (AbCam).

    Techniques: Incubation, Staining, Cholesterol Assay, Microscopy

    ApoER2 is colocalized with neuron-specific class III-tubulin (TUJ1) + -PCNs; ApoE2 treatment increases cholesterol uptake/redistribution in C57BL/6-PCNs. ( A ) PCNs in vehicle-control group; ( B ) PCNs in ApoE2 (4 µg/mL) treatment group; ( C ) Filipin III stained PCNs with or without ApoE2-treatment (arrow points cholesterol particles in soma of PCNs, arrow-head shows cholesterol particles in dendrites of PCNs).

    Journal: International Journal of Molecular Sciences

    Article Title: Administration of Downstream ApoE Attenuates the Adverse Effect of Brain ABCA1 Deficiency on Stroke

    doi: 10.3390/ijms19113368

    Figure Lengend Snippet: ApoER2 is colocalized with neuron-specific class III-tubulin (TUJ1) + -PCNs; ApoE2 treatment increases cholesterol uptake/redistribution in C57BL/6-PCNs. ( A ) PCNs in vehicle-control group; ( B ) PCNs in ApoE2 (4 µg/mL) treatment group; ( C ) Filipin III stained PCNs with or without ApoE2-treatment (arrow points cholesterol particles in soma of PCNs, arrow-head shows cholesterol particles in dendrites of PCNs).

    Article Snippet: The intracellular free cholesterol particles were monitored by Filipin III staining using a cell-based cholesterol assay kit (ab133116, Abcam, Cambridge, UK).

    Techniques: Staining

    Npc1 -/- mice liver and NPC cells show increased MLN64 expression. (A) To detect protein expression 30 μg of protein from liver homogenates were subjected to SDS-PAGE and western blotting for MLN64. ε-COP was used as a loading control. The Figure shows a western blot representative image, n=5. (B) Western blots bands intensity quantification. (C) Real time PCR analysis of MLN64 mRNA from mouse liver, n=4. (D) 30 μg of protein of CHO-WT and CHO-NPC cell extracts were analyzed by western blot against MLN64 and ε-COP. A representative image is shown, n=3. (E) Western blots bands intensity quantification. (F) MLN64 immunofluorescence analysis (Top). CHO-WT and CHO-NPC cells were immunostained with an anti-MLN64 antibody. Filipin staining (Bottom). CHO-WT and CHO-NPC cells were fixed, and cholesterol accumulation was detected by filipin staining. Scale bar: 20 µm (G) Graph shows quantifications of fluorescence. * Indicates statistically significant differences (p

    Journal: Redox Biology

    Article Title: MLN64 induces mitochondrial dysfunction associated with increased mitochondrial cholesterol content

    doi: 10.1016/j.redox.2017.02.024

    Figure Lengend Snippet: Npc1 -/- mice liver and NPC cells show increased MLN64 expression. (A) To detect protein expression 30 μg of protein from liver homogenates were subjected to SDS-PAGE and western blotting for MLN64. ε-COP was used as a loading control. The Figure shows a western blot representative image, n=5. (B) Western blots bands intensity quantification. (C) Real time PCR analysis of MLN64 mRNA from mouse liver, n=4. (D) 30 μg of protein of CHO-WT and CHO-NPC cell extracts were analyzed by western blot against MLN64 and ε-COP. A representative image is shown, n=3. (E) Western blots bands intensity quantification. (F) MLN64 immunofluorescence analysis (Top). CHO-WT and CHO-NPC cells were immunostained with an anti-MLN64 antibody. Filipin staining (Bottom). CHO-WT and CHO-NPC cells were fixed, and cholesterol accumulation was detected by filipin staining. Scale bar: 20 µm (G) Graph shows quantifications of fluorescence. * Indicates statistically significant differences (p

    Article Snippet: Cells were incubated for 2 h with 25 μg/ml filipin, rinsed with PBS, followed by incubation with primary antibodies, mouse anti-cytochrome C antibody (1:200, ab13575, Abcam) and rabbit anti-MLN64 polyclonal antibody (1:500), rinsed with PBS, and incubated for 2 h with secondary antibodies (1:1000).

    Techniques: Mouse Assay, Expressing, SDS Page, Western Blot, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Fluorescence

    MLN64 overexpression increases mitochondrial cholesterol and decreases mitochondrial glutathione and ATPase activity in cultured hepatocytes (A) Immunofluorescence and filipin staining analysis of Ad.E1Δ and Ad.MLN64 HepG2 cells stained with anti-MLN64 (MLN64, red), anti-cytochrome c (Cyt-C, green) and filipin (cyan) 48 h after infection. Colocalization of Cyt-C and filipin indicating of mitochondrial cholesterol is depicted (white). Merge and zoom of the merge. Scale bar: 20 µm. (B) The graph shows the Pearson’s coefficient that measures the percentage of colocalization of filipin and Cyt-C. The photographs shown are representative confocal images, (n=3). (C) Mitochondrial glutathione content. Mitochondria were isolated by Percoll density ultracentrifugation from Ad.E1Δ and Ad.MLN64 HepG2 cells 48 h after infection. Glutathione in mitochondria was determined as described in the Materials and Methods section, (n=4). (D) ATPase activity in mitochondrial fraction from HepG2 cells (n=4) was quantified by measuring the hydrolysis rate of ATP. *Indicates statistically significant differences (p

    Journal: Redox Biology

    Article Title: MLN64 induces mitochondrial dysfunction associated with increased mitochondrial cholesterol content

    doi: 10.1016/j.redox.2017.02.024

    Figure Lengend Snippet: MLN64 overexpression increases mitochondrial cholesterol and decreases mitochondrial glutathione and ATPase activity in cultured hepatocytes (A) Immunofluorescence and filipin staining analysis of Ad.E1Δ and Ad.MLN64 HepG2 cells stained with anti-MLN64 (MLN64, red), anti-cytochrome c (Cyt-C, green) and filipin (cyan) 48 h after infection. Colocalization of Cyt-C and filipin indicating of mitochondrial cholesterol is depicted (white). Merge and zoom of the merge. Scale bar: 20 µm. (B) The graph shows the Pearson’s coefficient that measures the percentage of colocalization of filipin and Cyt-C. The photographs shown are representative confocal images, (n=3). (C) Mitochondrial glutathione content. Mitochondria were isolated by Percoll density ultracentrifugation from Ad.E1Δ and Ad.MLN64 HepG2 cells 48 h after infection. Glutathione in mitochondria was determined as described in the Materials and Methods section, (n=4). (D) ATPase activity in mitochondrial fraction from HepG2 cells (n=4) was quantified by measuring the hydrolysis rate of ATP. *Indicates statistically significant differences (p

    Article Snippet: Cells were incubated for 2 h with 25 μg/ml filipin, rinsed with PBS, followed by incubation with primary antibodies, mouse anti-cytochrome C antibody (1:200, ab13575, Abcam) and rabbit anti-MLN64 polyclonal antibody (1:500), rinsed with PBS, and incubated for 2 h with secondary antibodies (1:1000).

    Techniques: Over Expression, Activity Assay, Cell Culture, Immunofluorescence, Staining, Infection, Isolation