Structured Review

Polysciences inc filipin solution
Correction of the cholesterol accumulation phenotype in a complementation test using different NPC1 variants. ( a ) Sanger sequencing results show the insertion of the desired point mutations into the vector containing NPC1 cDNA. ( b ) <t>Filipin</t> staining. WT NPC1 and different variants were investigated for cholesterol clearance in CHO Npc1 cells 12 h post transfection. ( c ) Quantification of the positive cholesterol signal. Values are shown as mean ± SD. Asterisks indicate statistical significance: ** p
Filipin Solution, supplied by Polysciences inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/filipin solution/product/Polysciences inc
Average 95 stars, based on 1 article reviews
Price from $9.99 to $1999.99
filipin solution - by Bioz Stars, 2020-04
95/100 stars

Images

1) Product Images from "Determination of the Pathological Features of NPC1 Variants in a Cellular Complementation Test"

Article Title: Determination of the Pathological Features of NPC1 Variants in a Cellular Complementation Test

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20205185

Correction of the cholesterol accumulation phenotype in a complementation test using different NPC1 variants. ( a ) Sanger sequencing results show the insertion of the desired point mutations into the vector containing NPC1 cDNA. ( b ) Filipin staining. WT NPC1 and different variants were investigated for cholesterol clearance in CHO Npc1 cells 12 h post transfection. ( c ) Quantification of the positive cholesterol signal. Values are shown as mean ± SD. Asterisks indicate statistical significance: ** p
Figure Legend Snippet: Correction of the cholesterol accumulation phenotype in a complementation test using different NPC1 variants. ( a ) Sanger sequencing results show the insertion of the desired point mutations into the vector containing NPC1 cDNA. ( b ) Filipin staining. WT NPC1 and different variants were investigated for cholesterol clearance in CHO Npc1 cells 12 h post transfection. ( c ) Quantification of the positive cholesterol signal. Values are shown as mean ± SD. Asterisks indicate statistical significance: ** p

Techniques Used: Sequencing, Plasmid Preparation, Staining, Transfection

Reduced cholesterol clearance in NPC1 -deficient Chinese hamster ovary (CHO) cells expressing variant p.Ile1061Thr NPC1 . CHO NPC1 cells develop a phenotype of cholesterol accumulation in lysosome-like storage organelles (LSOs). This phenotype can be corrected by gene transfer of wild type NPC1 cDNA using liposome-mediated cell transfection. ( a ) Fluorescence microscopic images. The blue staining visualizes cellular filipin-bound cholesterol. The arrowheads indicate LSOs. The green signal shows positively transfected cells that could be used for filipin quantification by either indicating GFP signal (control vector transfection) or NPC1-GFP fusion protein signal. ( b ) Quantification of the positive cholesterol signal. The y-axis shows the relative fluorescence intensity (RFI) signal as arbitrary units. Values are shown as mean ± SD. Asterisks indicate statistical significance: *** p
Figure Legend Snippet: Reduced cholesterol clearance in NPC1 -deficient Chinese hamster ovary (CHO) cells expressing variant p.Ile1061Thr NPC1 . CHO NPC1 cells develop a phenotype of cholesterol accumulation in lysosome-like storage organelles (LSOs). This phenotype can be corrected by gene transfer of wild type NPC1 cDNA using liposome-mediated cell transfection. ( a ) Fluorescence microscopic images. The blue staining visualizes cellular filipin-bound cholesterol. The arrowheads indicate LSOs. The green signal shows positively transfected cells that could be used for filipin quantification by either indicating GFP signal (control vector transfection) or NPC1-GFP fusion protein signal. ( b ) Quantification of the positive cholesterol signal. The y-axis shows the relative fluorescence intensity (RFI) signal as arbitrary units. Values are shown as mean ± SD. Asterisks indicate statistical significance: *** p

Techniques Used: Expressing, Variant Assay, Transfection, Fluorescence, Staining, Plasmid Preparation

Cholesterol elimination by the p.Ile1061Thr variant can be assisted by 25-hydroxycholesterol. The black bars indicate the filipin signal of untreated or 5 µM 25-hydroxycholesterol treated CHO NPC1 cells transfected with either pCMV6 control vector or WT NPC1-GFP vector 24 h post-transfection. Values are shown as mean ± SD. Asterisks indicate statistical significance: * p
Figure Legend Snippet: Cholesterol elimination by the p.Ile1061Thr variant can be assisted by 25-hydroxycholesterol. The black bars indicate the filipin signal of untreated or 5 µM 25-hydroxycholesterol treated CHO NPC1 cells transfected with either pCMV6 control vector or WT NPC1-GFP vector 24 h post-transfection. Values are shown as mean ± SD. Asterisks indicate statistical significance: * p

Techniques Used: Variant Assay, Transfection, Plasmid Preparation

2) Product Images from "Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice [S]"

Article Title: Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice [S]

Journal: Journal of Lipid Research

doi: 10.1194/jlr.M048629

Effect of LCAT on RBC and platelet counts and FC content. A: RBC counts of mice on chow or Western diet (7 months). B: RBC FC content, as determined by filipin staining of mice on chow or Western diet (7 months). C: Platelet counts of mice on chow or
Figure Legend Snippet: Effect of LCAT on RBC and platelet counts and FC content. A: RBC counts of mice on chow or Western diet (7 months). B: RBC FC content, as determined by filipin staining of mice on chow or Western diet (7 months). C: Platelet counts of mice on chow or

Techniques Used: Mouse Assay, Western Blot, Staining

Effect of LCAT on FC in the aortic sinus. A: Amount of FC in the intima of the aortic sinus was assessed by filipin staining. B–E: Representative images of cholesterol crystals detected by polarized light and the amount of FC detected by filipin
Figure Legend Snippet: Effect of LCAT on FC in the aortic sinus. A: Amount of FC in the intima of the aortic sinus was assessed by filipin staining. B–E: Representative images of cholesterol crystals detected by polarized light and the amount of FC detected by filipin

Techniques Used: Staining

Related Articles

Flow Cytometry:

Article Title: Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice [S]
Article Snippet: Following fixation, cells were washed one time with PBS and resuspended in 500 μl of a 120 μg/ml filipin solution (Polysciences, Inc., Warrington, PA) supplemented with 2% dextran. .. Cells were kept on ice until they could be analyzed by flow cytometry, using an LSRFortessa or FACsARIA (BD Biosciences).

Fluorescence:

Article Title: Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice [S]
Article Snippet: Paragraph title: Fluorescence-activated cell sorting analysis of platelets and erythrocytes ... Following fixation, cells were washed one time with PBS and resuspended in 500 μl of a 120 μg/ml filipin solution (Polysciences, Inc., Warrington, PA) supplemented with 2% dextran.

Article Title: Determination of the Pathological Features of NPC1 Variants in a Cellular Complementation Test
Article Snippet: Paragraph title: 3.2.6. Filipin Staining and Fluorescence Microscopy ... Prior to transfection with Lipofectamin 2000 the cells were allowed to adhere for 24 h. At 12 and 24 h after the transfection, the CHO cells were fixed using 4% PFA in PBS at pH 7.4 for 15 min followed by washing with PBS and incubation with filipin solution (0.5 mg/mL in PBS; Polysciences, Warrington, PA, USA) at room temperature in the dark for 45 min. After extended washing in PBS in a dark chamber, the coverslips were mounted with Mowiol mounting medium (Mowiol, #81831, Sigma) and stored at 4 °C until the analysis.

Cytometry:

Article Title: Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice [S]
Article Snippet: Following fixation, cells were washed one time with PBS and resuspended in 500 μl of a 120 μg/ml filipin solution (Polysciences, Inc., Warrington, PA) supplemented with 2% dextran. .. Cells were kept on ice until they could be analyzed by flow cytometry, using an LSRFortessa or FACsARIA (BD Biosciences).

Microscopy:

Article Title: Determination of the Pathological Features of NPC1 Variants in a Cellular Complementation Test
Article Snippet: Paragraph title: 3.2.6. Filipin Staining and Fluorescence Microscopy ... Prior to transfection with Lipofectamin 2000 the cells were allowed to adhere for 24 h. At 12 and 24 h after the transfection, the CHO cells were fixed using 4% PFA in PBS at pH 7.4 for 15 min followed by washing with PBS and incubation with filipin solution (0.5 mg/mL in PBS; Polysciences, Warrington, PA, USA) at room temperature in the dark for 45 min. After extended washing in PBS in a dark chamber, the coverslips were mounted with Mowiol mounting medium (Mowiol, #81831, Sigma) and stored at 4 °C until the analysis.

Article Title: Tricyclic pyrone compounds prevent aggregation and reverse cellular phenotypes caused by expression of mutant huntingtin protein in striatal neurons
Article Snippet: Coverslips were washed 3 times with PBS, incubated 30 min with glycine (75 mg in 100 ml of PBS), and filipin solution (100 μg/ml, Polysciences, Inc., Warrington, PA) was applied for 30 min at room temperature. .. Cells were washed in PBS and immediately observed under the microscope (LSM 510, C. Zeiss) using 100× magnification.

Mouse Assay:

Article Title: Tricyclic pyrone compounds prevent aggregation and reverse cellular phenotypes caused by expression of mutant huntingtin protein in striatal neurons
Article Snippet: Filipin staining in neurons Striatal neurons from control and HD72 mice were plated on poly-L-ornithine covered glass coverslips in 6-well culture dishes in the serum-containing medium. .. Coverslips were washed 3 times with PBS, incubated 30 min with glycine (75 mg in 100 ml of PBS), and filipin solution (100 μg/ml, Polysciences, Inc., Warrington, PA) was applied for 30 min at room temperature.

Concentration Assay:

Article Title: Determination of the Pathological Features of NPC1 Variants in a Cellular Complementation Test
Article Snippet: Filipin Staining and Fluorescence Microscopy CHO cells were seeded on coverslips placed in the wells of a 24-well dish at a concentration of 0.26 × 106 /mL. .. Prior to transfection with Lipofectamin 2000 the cells were allowed to adhere for 24 h. At 12 and 24 h after the transfection, the CHO cells were fixed using 4% PFA in PBS at pH 7.4 for 15 min followed by washing with PBS and incubation with filipin solution (0.5 mg/mL in PBS; Polysciences, Warrington, PA, USA) at room temperature in the dark for 45 min. After extended washing in PBS in a dark chamber, the coverslips were mounted with Mowiol mounting medium (Mowiol, #81831, Sigma) and stored at 4 °C until the analysis.

Incubation:

Article Title: Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice [S]
Article Snippet: Cells were resuspended in 80 μl of antibody solution and incubated at room temperature for 20 min. After incubation, cells were fixed with 4% paraformaldehyde for 1 h at 4°C. .. Following fixation, cells were washed one time with PBS and resuspended in 500 μl of a 120 μg/ml filipin solution (Polysciences, Inc., Warrington, PA) supplemented with 2% dextran.

Article Title: Determination of the Pathological Features of NPC1 Variants in a Cellular Complementation Test
Article Snippet: .. Prior to transfection with Lipofectamin 2000 the cells were allowed to adhere for 24 h. At 12 and 24 h after the transfection, the CHO cells were fixed using 4% PFA in PBS at pH 7.4 for 15 min followed by washing with PBS and incubation with filipin solution (0.5 mg/mL in PBS; Polysciences, Warrington, PA, USA) at room temperature in the dark for 45 min. After extended washing in PBS in a dark chamber, the coverslips were mounted with Mowiol mounting medium (Mowiol, #81831, Sigma) and stored at 4 °C until the analysis. .. Imaging was carried out using a digital compact microscope (BZ-8000; Keyence, Neu-Isenburg, Germany) with a 40X/NA0.95 objective.

Article Title: Tricyclic pyrone compounds prevent aggregation and reverse cellular phenotypes caused by expression of mutant huntingtin protein in striatal neurons
Article Snippet: .. Coverslips were washed 3 times with PBS, incubated 30 min with glycine (75 mg in 100 ml of PBS), and filipin solution (100 μg/ml, Polysciences, Inc., Warrington, PA) was applied for 30 min at room temperature. .. Cells were washed in PBS and immediately observed under the microscope (LSM 510, C. Zeiss) using 100× magnification.

Transfection:

Article Title: Determination of the Pathological Features of NPC1 Variants in a Cellular Complementation Test
Article Snippet: .. Prior to transfection with Lipofectamin 2000 the cells were allowed to adhere for 24 h. At 12 and 24 h after the transfection, the CHO cells were fixed using 4% PFA in PBS at pH 7.4 for 15 min followed by washing with PBS and incubation with filipin solution (0.5 mg/mL in PBS; Polysciences, Warrington, PA, USA) at room temperature in the dark for 45 min. After extended washing in PBS in a dark chamber, the coverslips were mounted with Mowiol mounting medium (Mowiol, #81831, Sigma) and stored at 4 °C until the analysis. .. Imaging was carried out using a digital compact microscope (BZ-8000; Keyence, Neu-Isenburg, Germany) with a 40X/NA0.95 objective.

Imaging:

Article Title: Determination of the Pathological Features of NPC1 Variants in a Cellular Complementation Test
Article Snippet: Prior to transfection with Lipofectamin 2000 the cells were allowed to adhere for 24 h. At 12 and 24 h after the transfection, the CHO cells were fixed using 4% PFA in PBS at pH 7.4 for 15 min followed by washing with PBS and incubation with filipin solution (0.5 mg/mL in PBS; Polysciences, Warrington, PA, USA) at room temperature in the dark for 45 min. After extended washing in PBS in a dark chamber, the coverslips were mounted with Mowiol mounting medium (Mowiol, #81831, Sigma) and stored at 4 °C until the analysis. .. Imaging was carried out using a digital compact microscope (BZ-8000; Keyence, Neu-Isenburg, Germany) with a 40X/NA0.95 objective.

Mass Spectrometry:

Article Title: Determination of the Pathological Features of NPC1 Variants in a Cellular Complementation Test
Article Snippet: Prior to transfection with Lipofectamin 2000 the cells were allowed to adhere for 24 h. At 12 and 24 h after the transfection, the CHO cells were fixed using 4% PFA in PBS at pH 7.4 for 15 min followed by washing with PBS and incubation with filipin solution (0.5 mg/mL in PBS; Polysciences, Warrington, PA, USA) at room temperature in the dark for 45 min. After extended washing in PBS in a dark chamber, the coverslips were mounted with Mowiol mounting medium (Mowiol, #81831, Sigma) and stored at 4 °C until the analysis. .. Exposure time for filipin: 100 ms; exposure time for GFP: 25 ms. Only NPC1-GFP positive cells were included in cholesterol quantification.

Staining:

Article Title: Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice [S]
Article Snippet: Following fixation, cells were washed one time with PBS and resuspended in 500 μl of a 120 μg/ml filipin solution (Polysciences, Inc., Warrington, PA) supplemented with 2% dextran. .. To assess the level of reticulated platelets, cells were stained as previously described ( , ).

Article Title: Determination of the Pathological Features of NPC1 Variants in a Cellular Complementation Test
Article Snippet: Paragraph title: 3.2.6. Filipin Staining and Fluorescence Microscopy ... Prior to transfection with Lipofectamin 2000 the cells were allowed to adhere for 24 h. At 12 and 24 h after the transfection, the CHO cells were fixed using 4% PFA in PBS at pH 7.4 for 15 min followed by washing with PBS and incubation with filipin solution (0.5 mg/mL in PBS; Polysciences, Warrington, PA, USA) at room temperature in the dark for 45 min. After extended washing in PBS in a dark chamber, the coverslips were mounted with Mowiol mounting medium (Mowiol, #81831, Sigma) and stored at 4 °C until the analysis.

Article Title: Tricyclic pyrone compounds prevent aggregation and reverse cellular phenotypes caused by expression of mutant huntingtin protein in striatal neurons
Article Snippet: Paragraph title: Filipin staining in neurons ... Coverslips were washed 3 times with PBS, incubated 30 min with glycine (75 mg in 100 ml of PBS), and filipin solution (100 μg/ml, Polysciences, Inc., Warrington, PA) was applied for 30 min at room temperature.

FACS:

Article Title: Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice [S]
Article Snippet: Paragraph title: Fluorescence-activated cell sorting analysis of platelets and erythrocytes ... Following fixation, cells were washed one time with PBS and resuspended in 500 μl of a 120 μg/ml filipin solution (Polysciences, Inc., Warrington, PA) supplemented with 2% dextran.

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    Polysciences inc filipin staining solution
    Unesterified cholesterol accumulation. (A) Control and mutant larvae were stained with <t>filipin</t> to visualize unesterified cholesterol. Filipin-positive puncta were initially observed in mutant larvae at 2 dpf in the area of the yolk sac extension and became more numerous with a wider distribution in older larvae. Scale bars: 200 µm. (B) Embryos were injected with TopFluor-cholesterol at the 1-cell stage and imaged at 7 dpf. Puncta of accumulated cholesterol were observed in the npc1 m/m larvae. Scale bar: 1 mm.
    Filipin Staining Solution, supplied by Polysciences inc, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/filipin staining solution/product/Polysciences inc
    Average 94 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    filipin staining solution - by Bioz Stars, 2020-04
    94/100 stars
      Buy from Supplier

    95
    Polysciences inc filipin solution
    Correction of the cholesterol accumulation phenotype in a complementation test using different NPC1 variants. ( a ) Sanger sequencing results show the insertion of the desired point mutations into the vector containing NPC1 cDNA. ( b ) <t>Filipin</t> staining. WT NPC1 and different variants were investigated for cholesterol clearance in CHO Npc1 cells 12 h post transfection. ( c ) Quantification of the positive cholesterol signal. Values are shown as mean ± SD. Asterisks indicate statistical significance: ** p
    Filipin Solution, supplied by Polysciences inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/filipin solution/product/Polysciences inc
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    filipin solution - by Bioz Stars, 2020-04
    95/100 stars
      Buy from Supplier

    Image Search Results


    Unesterified cholesterol accumulation. (A) Control and mutant larvae were stained with filipin to visualize unesterified cholesterol. Filipin-positive puncta were initially observed in mutant larvae at 2 dpf in the area of the yolk sac extension and became more numerous with a wider distribution in older larvae. Scale bars: 200 µm. (B) Embryos were injected with TopFluor-cholesterol at the 1-cell stage and imaged at 7 dpf. Puncta of accumulated cholesterol were observed in the npc1 m/m larvae. Scale bar: 1 mm.

    Journal: Disease Models & Mechanisms

    Article Title: Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds

    doi: 10.1242/dmm.034165

    Figure Lengend Snippet: Unesterified cholesterol accumulation. (A) Control and mutant larvae were stained with filipin to visualize unesterified cholesterol. Filipin-positive puncta were initially observed in mutant larvae at 2 dpf in the area of the yolk sac extension and became more numerous with a wider distribution in older larvae. Scale bars: 200 µm. (B) Embryos were injected with TopFluor-cholesterol at the 1-cell stage and imaged at 7 dpf. Puncta of accumulated cholesterol were observed in the npc1 m/m larvae. Scale bar: 1 mm.

    Article Snippet: After extensive rinsing with 1× PBS, fixed embryos/larvae were then stained with filipin staining solution containing 0.5 mg/ml filipin (08707, Polysciences, Warrington, PA, USA) and 1% goat serum in 1× PBS for 2.5 h at room temperature.

    Techniques: Mutagenesis, Staining, Injection

    2HPβCD treatment reduces LysoTracker Red and filipin staining in npc1 m/m neuromasts. (A) Control and mutant larvae can be differentiated at 3 dpf by intense LysoTracker Red (Lyso R) staining of the olfactory placode (arrowheads) at 3 dpf. Scale bars: 100 μm. (B) Mutant larvae selected by Lyso-R-positive olfactory placode staining were treated for 3 days with vehicle (ddH 2 O) or 2.5 mM 2HPβCD starting at 3 dpf. Neuromast Lyso R staining was markedly decreased in 2HPβCD-treated npc1 m/m larvae. Scale bars: 1 mm. (C) Higher magnification view of Lyso R staining, corresponding to the boxed area in B. Scale bar: 100 μm. (D) Lyso R relative intensity was significantly reduced in npc1 m/m larvae treated with 2.5 mM 2HPβCD at 6 dpf. **** P

    Journal: Disease Models & Mechanisms

    Article Title: Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds

    doi: 10.1242/dmm.034165

    Figure Lengend Snippet: 2HPβCD treatment reduces LysoTracker Red and filipin staining in npc1 m/m neuromasts. (A) Control and mutant larvae can be differentiated at 3 dpf by intense LysoTracker Red (Lyso R) staining of the olfactory placode (arrowheads) at 3 dpf. Scale bars: 100 μm. (B) Mutant larvae selected by Lyso-R-positive olfactory placode staining were treated for 3 days with vehicle (ddH 2 O) or 2.5 mM 2HPβCD starting at 3 dpf. Neuromast Lyso R staining was markedly decreased in 2HPβCD-treated npc1 m/m larvae. Scale bars: 1 mm. (C) Higher magnification view of Lyso R staining, corresponding to the boxed area in B. Scale bar: 100 μm. (D) Lyso R relative intensity was significantly reduced in npc1 m/m larvae treated with 2.5 mM 2HPβCD at 6 dpf. **** P

    Article Snippet: After extensive rinsing with 1× PBS, fixed embryos/larvae were then stained with filipin staining solution containing 0.5 mg/ml filipin (08707, Polysciences, Warrington, PA, USA) and 1% goat serum in 1× PBS for 2.5 h at room temperature.

    Techniques: Staining, Mutagenesis

    Yolk syncytial layer accumulation of unesterified cholesterol. Plasmids expressing either EGFP or npc1 were injected into the yolk syncytial layer (YSL) of 3.5 hpf embryos. Filipin staining of 2 dpf embryos demonstrated reversal of unesterified cholesterol accumulation in the YSL of mutant larvae expressing npc1 . Scale bar: 200 μm.

    Journal: Disease Models & Mechanisms

    Article Title: Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds

    doi: 10.1242/dmm.034165

    Figure Lengend Snippet: Yolk syncytial layer accumulation of unesterified cholesterol. Plasmids expressing either EGFP or npc1 were injected into the yolk syncytial layer (YSL) of 3.5 hpf embryos. Filipin staining of 2 dpf embryos demonstrated reversal of unesterified cholesterol accumulation in the YSL of mutant larvae expressing npc1 . Scale bar: 200 μm.

    Article Snippet: After extensive rinsing with 1× PBS, fixed embryos/larvae were then stained with filipin staining solution containing 0.5 mg/ml filipin (08707, Polysciences, Warrington, PA, USA) and 1% goat serum in 1× PBS for 2.5 h at room temperature.

    Techniques: Expressing, Injection, Staining, Mutagenesis

    Unesterified cholesterol accumulation. (A) Control and mutant larvae were stained with filipin to visualize unesterified cholesterol. Filipin-positive puncta were initially observed in mutant larvae at 2 dpf in the area of the yolk sac extension and became more numerous with a wider distribution in older larvae. Scale bars: 200 µm. (B) Embryos were injected with TopFluor-cholesterol at the 1-cell stage and imaged at 7 dpf. Puncta of accumulated cholesterol were observed in the npc1 m/m larvae. Scale bar: 1 mm.

    Journal: Disease Models & Mechanisms

    Article Title: Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds

    doi: 10.1242/dmm.034165

    Figure Lengend Snippet: Unesterified cholesterol accumulation. (A) Control and mutant larvae were stained with filipin to visualize unesterified cholesterol. Filipin-positive puncta were initially observed in mutant larvae at 2 dpf in the area of the yolk sac extension and became more numerous with a wider distribution in older larvae. Scale bars: 200 µm. (B) Embryos were injected with TopFluor-cholesterol at the 1-cell stage and imaged at 7 dpf. Puncta of accumulated cholesterol were observed in the npc1 m/m larvae. Scale bar: 1 mm.

    Article Snippet: After extensive rinsing with 1× PBS, fixed embryos/larvae were then stained with filipin staining solution containing 0.5 mg/ml filipin (08707, Polysciences, Warrington, PA, USA) and 1% goat serum in 1× PBS for 2.5 h at room temperature.

    Techniques: Mutagenesis, Staining, Injection

    2HPβCD treatment reduces LysoTracker Red and filipin staining in npc1 m/m neuromasts. (A) Control and mutant larvae can be differentiated at 3 dpf by intense LysoTracker Red (Lyso R) staining of the olfactory placode (arrowheads) at 3 dpf. Scale bars: 100 μm. (B) Mutant larvae selected by Lyso-R-positive olfactory placode staining were treated for 3 days with vehicle (ddH 2 O) or 2.5 mM 2HPβCD starting at 3 dpf. Neuromast Lyso R staining was markedly decreased in 2HPβCD-treated npc1 m/m larvae. Scale bars: 1 mm. (C) Higher magnification view of Lyso R staining, corresponding to the boxed area in B. Scale bar: 100 μm. (D) Lyso R relative intensity was significantly reduced in npc1 m/m larvae treated with 2.5 mM 2HPβCD at 6 dpf. **** P

    Journal: Disease Models & Mechanisms

    Article Title: Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds

    doi: 10.1242/dmm.034165

    Figure Lengend Snippet: 2HPβCD treatment reduces LysoTracker Red and filipin staining in npc1 m/m neuromasts. (A) Control and mutant larvae can be differentiated at 3 dpf by intense LysoTracker Red (Lyso R) staining of the olfactory placode (arrowheads) at 3 dpf. Scale bars: 100 μm. (B) Mutant larvae selected by Lyso-R-positive olfactory placode staining were treated for 3 days with vehicle (ddH 2 O) or 2.5 mM 2HPβCD starting at 3 dpf. Neuromast Lyso R staining was markedly decreased in 2HPβCD-treated npc1 m/m larvae. Scale bars: 1 mm. (C) Higher magnification view of Lyso R staining, corresponding to the boxed area in B. Scale bar: 100 μm. (D) Lyso R relative intensity was significantly reduced in npc1 m/m larvae treated with 2.5 mM 2HPβCD at 6 dpf. **** P

    Article Snippet: After extensive rinsing with 1× PBS, fixed embryos/larvae were then stained with filipin staining solution containing 0.5 mg/ml filipin (08707, Polysciences, Warrington, PA, USA) and 1% goat serum in 1× PBS for 2.5 h at room temperature.

    Techniques: Staining, Mutagenesis

    Yolk syncytial layer accumulation of unesterified cholesterol. Plasmids expressing either EGFP or npc1 were injected into the yolk syncytial layer (YSL) of 3.5 hpf embryos. Filipin staining of 2 dpf embryos demonstrated reversal of unesterified cholesterol accumulation in the YSL of mutant larvae expressing npc1 . Scale bar: 200 μm.

    Journal: Disease Models & Mechanisms

    Article Title: Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds

    doi: 10.1242/dmm.034165

    Figure Lengend Snippet: Yolk syncytial layer accumulation of unesterified cholesterol. Plasmids expressing either EGFP or npc1 were injected into the yolk syncytial layer (YSL) of 3.5 hpf embryos. Filipin staining of 2 dpf embryos demonstrated reversal of unesterified cholesterol accumulation in the YSL of mutant larvae expressing npc1 . Scale bar: 200 μm.

    Article Snippet: After extensive rinsing with 1× PBS, fixed embryos/larvae were then stained with filipin staining solution containing 0.5 mg/ml filipin (08707, Polysciences, Warrington, PA, USA) and 1% goat serum in 1× PBS for 2.5 h at room temperature.

    Techniques: Expressing, Injection, Staining, Mutagenesis

    Correction of the cholesterol accumulation phenotype in a complementation test using different NPC1 variants. ( a ) Sanger sequencing results show the insertion of the desired point mutations into the vector containing NPC1 cDNA. ( b ) Filipin staining. WT NPC1 and different variants were investigated for cholesterol clearance in CHO Npc1 cells 12 h post transfection. ( c ) Quantification of the positive cholesterol signal. Values are shown as mean ± SD. Asterisks indicate statistical significance: ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Determination of the Pathological Features of NPC1 Variants in a Cellular Complementation Test

    doi: 10.3390/ijms20205185

    Figure Lengend Snippet: Correction of the cholesterol accumulation phenotype in a complementation test using different NPC1 variants. ( a ) Sanger sequencing results show the insertion of the desired point mutations into the vector containing NPC1 cDNA. ( b ) Filipin staining. WT NPC1 and different variants were investigated for cholesterol clearance in CHO Npc1 cells 12 h post transfection. ( c ) Quantification of the positive cholesterol signal. Values are shown as mean ± SD. Asterisks indicate statistical significance: ** p

    Article Snippet: Prior to transfection with Lipofectamin 2000 the cells were allowed to adhere for 24 h. At 12 and 24 h after the transfection, the CHO cells were fixed using 4% PFA in PBS at pH 7.4 for 15 min followed by washing with PBS and incubation with filipin solution (0.5 mg/mL in PBS; Polysciences, Warrington, PA, USA) at room temperature in the dark for 45 min. After extended washing in PBS in a dark chamber, the coverslips were mounted with Mowiol mounting medium (Mowiol, #81831, Sigma) and stored at 4 °C until the analysis.

    Techniques: Sequencing, Plasmid Preparation, Staining, Transfection

    Reduced cholesterol clearance in NPC1 -deficient Chinese hamster ovary (CHO) cells expressing variant p.Ile1061Thr NPC1 . CHO NPC1 cells develop a phenotype of cholesterol accumulation in lysosome-like storage organelles (LSOs). This phenotype can be corrected by gene transfer of wild type NPC1 cDNA using liposome-mediated cell transfection. ( a ) Fluorescence microscopic images. The blue staining visualizes cellular filipin-bound cholesterol. The arrowheads indicate LSOs. The green signal shows positively transfected cells that could be used for filipin quantification by either indicating GFP signal (control vector transfection) or NPC1-GFP fusion protein signal. ( b ) Quantification of the positive cholesterol signal. The y-axis shows the relative fluorescence intensity (RFI) signal as arbitrary units. Values are shown as mean ± SD. Asterisks indicate statistical significance: *** p

    Journal: International Journal of Molecular Sciences

    Article Title: Determination of the Pathological Features of NPC1 Variants in a Cellular Complementation Test

    doi: 10.3390/ijms20205185

    Figure Lengend Snippet: Reduced cholesterol clearance in NPC1 -deficient Chinese hamster ovary (CHO) cells expressing variant p.Ile1061Thr NPC1 . CHO NPC1 cells develop a phenotype of cholesterol accumulation in lysosome-like storage organelles (LSOs). This phenotype can be corrected by gene transfer of wild type NPC1 cDNA using liposome-mediated cell transfection. ( a ) Fluorescence microscopic images. The blue staining visualizes cellular filipin-bound cholesterol. The arrowheads indicate LSOs. The green signal shows positively transfected cells that could be used for filipin quantification by either indicating GFP signal (control vector transfection) or NPC1-GFP fusion protein signal. ( b ) Quantification of the positive cholesterol signal. The y-axis shows the relative fluorescence intensity (RFI) signal as arbitrary units. Values are shown as mean ± SD. Asterisks indicate statistical significance: *** p

    Article Snippet: Prior to transfection with Lipofectamin 2000 the cells were allowed to adhere for 24 h. At 12 and 24 h after the transfection, the CHO cells were fixed using 4% PFA in PBS at pH 7.4 for 15 min followed by washing with PBS and incubation with filipin solution (0.5 mg/mL in PBS; Polysciences, Warrington, PA, USA) at room temperature in the dark for 45 min. After extended washing in PBS in a dark chamber, the coverslips were mounted with Mowiol mounting medium (Mowiol, #81831, Sigma) and stored at 4 °C until the analysis.

    Techniques: Expressing, Variant Assay, Transfection, Fluorescence, Staining, Plasmid Preparation

    Cholesterol elimination by the p.Ile1061Thr variant can be assisted by 25-hydroxycholesterol. The black bars indicate the filipin signal of untreated or 5 µM 25-hydroxycholesterol treated CHO NPC1 cells transfected with either pCMV6 control vector or WT NPC1-GFP vector 24 h post-transfection. Values are shown as mean ± SD. Asterisks indicate statistical significance: * p

    Journal: International Journal of Molecular Sciences

    Article Title: Determination of the Pathological Features of NPC1 Variants in a Cellular Complementation Test

    doi: 10.3390/ijms20205185

    Figure Lengend Snippet: Cholesterol elimination by the p.Ile1061Thr variant can be assisted by 25-hydroxycholesterol. The black bars indicate the filipin signal of untreated or 5 µM 25-hydroxycholesterol treated CHO NPC1 cells transfected with either pCMV6 control vector or WT NPC1-GFP vector 24 h post-transfection. Values are shown as mean ± SD. Asterisks indicate statistical significance: * p

    Article Snippet: Prior to transfection with Lipofectamin 2000 the cells were allowed to adhere for 24 h. At 12 and 24 h after the transfection, the CHO cells were fixed using 4% PFA in PBS at pH 7.4 for 15 min followed by washing with PBS and incubation with filipin solution (0.5 mg/mL in PBS; Polysciences, Warrington, PA, USA) at room temperature in the dark for 45 min. After extended washing in PBS in a dark chamber, the coverslips were mounted with Mowiol mounting medium (Mowiol, #81831, Sigma) and stored at 4 °C until the analysis.

    Techniques: Variant Assay, Transfection, Plasmid Preparation

    Effect of LCAT on RBC and platelet counts and FC content. A: RBC counts of mice on chow or Western diet (7 months). B: RBC FC content, as determined by filipin staining of mice on chow or Western diet (7 months). C: Platelet counts of mice on chow or

    Journal: Journal of Lipid Research

    Article Title: Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice [S]

    doi: 10.1194/jlr.M048629

    Figure Lengend Snippet: Effect of LCAT on RBC and platelet counts and FC content. A: RBC counts of mice on chow or Western diet (7 months). B: RBC FC content, as determined by filipin staining of mice on chow or Western diet (7 months). C: Platelet counts of mice on chow or

    Article Snippet: Following fixation, cells were washed one time with PBS and resuspended in 500 μl of a 120 μg/ml filipin solution (Polysciences, Inc., Warrington, PA) supplemented with 2% dextran.

    Techniques: Mouse Assay, Western Blot, Staining

    Effect of LCAT on FC in the aortic sinus. A: Amount of FC in the intima of the aortic sinus was assessed by filipin staining. B–E: Representative images of cholesterol crystals detected by polarized light and the amount of FC detected by filipin

    Journal: Journal of Lipid Research

    Article Title: Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice [S]

    doi: 10.1194/jlr.M048629

    Figure Lengend Snippet: Effect of LCAT on FC in the aortic sinus. A: Amount of FC in the intima of the aortic sinus was assessed by filipin staining. B–E: Representative images of cholesterol crystals detected by polarized light and the amount of FC detected by filipin

    Article Snippet: Following fixation, cells were washed one time with PBS and resuspended in 500 μl of a 120 μg/ml filipin solution (Polysciences, Inc., Warrington, PA) supplemented with 2% dextran.

    Techniques: Staining