fitc conjugated spc  (Bioss)

Journal: Antioxidants

Article Title: By-Product Extracts from Castanea sativa Counteract Hallmarks of Neuroinflammation in a Microglial Model

doi: 10.3390/antiox12040808

Figure Lengend Snippet: Primer used for Real-time qPCR.

Article Snippet: Rabbit anti-TLR4 polyclonal FITC-conjugated antibody was purchased from StressMarq.

Techniques: Sequencing

Journal: Antioxidants

Article Title: By-Product Extracts from Castanea sativa Counteract Hallmarks of Neuroinflammation in a Microglial Model

doi: 10.3390/antiox12040808

Figure Lengend Snippet: Cell surface level of TLR4 in BV-2 cells treated with chestnut leaf or spiny bur extracts and LPS. BV-2 cells were pre-treated (or not) with spiny bur (SB) or leaf (L) extracts (0.5 mg/mL) for 3 h, incubated with or without LPS (0.5 μg/mL) for a total of 24 h, then subjected to flow cytometric analysis of the immunostaining for TLR4 receptor. ( a ) Representative plots of untreated BV-2 cells ( b ) Representative plots of BV-2 cells not activated with LPS ( c ) Representative plots of LPS-stimulated BV-2 cells ( d ) Relative quantification is expressed as MFI, median fluorescence intensity. Results are expressed as means ± SEM of three independent experiments. Statistical analysis was performed by Fisher’s LSD test following one-way ANOVA. ° p < 0.05 significantly different from control cells; * p < 0.05 significantly different from LPS-treated cells.

Article Snippet: Rabbit anti-TLR4 polyclonal FITC-conjugated antibody was purchased from StressMarq.

Techniques: Incubation, Immunostaining, Fluorescence, Control

Journal: Antioxidants

Article Title: By-Product Extracts from Castanea sativa Counteract Hallmarks of Neuroinflammation in a Microglial Model

doi: 10.3390/antiox12040808

Figure Lengend Snippet: RT-PCR analysis of TLR4 gene expression in BV-2 cells treated with chestnut extracts. BV-2 cells were treated with spiny bur or leaf extracts (0.5 mg/mL) for 3 h, then stimulated (or not) with LPS (0.5 µg/mL) for a total of 24 h. At the end of incubation, RNA was extracted from cells and samples were subjected to RT-PCR analysis using a specific primer for TLR4, as explained in the Materials and Methods section. The relative mRNA content of TLR4 was normalised to ( a ) untreated control and ( b ) untreated LPS control. Relative quantification is obtained as reported in the Materials and Methods section; results are expressed as means ± SEM of three independent experiments. Statistical analysis was performed by Fisher’s LSD test following one-way ANOVA. ° p < 0.05 significantly different from control cells; * p < 0.05 significantly different from LPS-treated cells.

Article Snippet: Rabbit anti-TLR4 polyclonal FITC-conjugated antibody was purchased from StressMarq.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation, Control

Journal: Antioxidants

Article Title: By-Product Extracts from Castanea sativa Counteract Hallmarks of Neuroinflammation in a Microglial Model

doi: 10.3390/antiox12040808

Figure Lengend Snippet: RT-PCR analysis of TLR4 and CD14 gene expression in BV-2 cells treated with different chestnut leaf extract fractions. BV-2 cells were treated for 3 h with 50 μg/mL of fractions at different polarity (L Fr.), then stimulated with 0.5 μg/mL LPS for a total of 24 h. At the end of incubation, RNA was extracted from cells and samples were subjected to RT-PCR analysis using a specific primer for TLR4 ( a ) and CD14 ( b ), as reported in the Materials and Methods section. The relative mRNA contents of TLR4 and CD14 were normalised to LPS-treated cells (red bars). Relative quantification is obtained as described in the Materials and Methods section, and results are expressed as means ± SEM of three independent experiments. Statistical analysis was performed by Fisher’s LSD test following one-way ANOVA. * p < 0.05 significantly different from LPS-treated cells.

Article Snippet: Rabbit anti-TLR4 polyclonal FITC-conjugated antibody was purchased from StressMarq.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation

Journal: Antioxidants & Redox Signaling

Article Title: Renal NOXA1/NOX1 Signaling Regulates Epithelial Sodium Channel and Sodium Retention in Angiotensin II-induced Hypertension

doi: 10.1089/ars.2021.0047

Figure Lengend Snippet: NOXA1 protein expression in kidney is regulated by Ang II. (A) Western blot analysis and quantification of NOXA1 expression in kidney lysates after 1 and 14 days of Ang II treatment. Data are NOXA1 protein fold change relative to untreated control adjusted for β-tubulin levels (mean ± SEM). (B) Western blot analysis and relative expression quantification of protein extracts from isolated PCT, TAL, and CD. (C) Representative immunofluorescence images and quantification of immunoreactive NOX1 and AQP1 (colocalized to PCT), NKCC2 (colocalized to TAL), or AQP2 (colocalized to CD) in wild-type kidney frozen sections (mean ± SEM). High magnification insets (yellow rectangle) show NOX1 expression in epithelial cell. Scale is 100 μm. (D) Representative immunofluorescence images and quantification stained for immunoreactive NOXA1 and AQP1 (colocalized to PCT), NKCC2 (colocalized to TAL), or AQP2 (colocalized to CD) in wild-type kidney frozen sections (mean ± SEM). High magnification insets (yellow rectangle) show NOXA1 expression in epithelial cell. Scale is 100 μm. Ang II, angiotensin II; AQP1, aquaporin 1; CD, collecting ducts; NKCC2, Na-K-Cl cotransporter 2; NOX, NADPH oxidase; NOXA1, NOX activator 1; PCT, proximal convoluted tubules; TAL, thick ascending limb.

Article Snippet: Frozen kidney sections were fixed in acetone, permeabilized in 0.1% Triton X-100, and immunofluorescent staining was performed using antibodies against NOXA1 (Ab199; a gift from Dr. Ralf Brandes, Institut für Kardiovaskuläre Physiologie, Goethe-Universität, Frankfurt am Main, Germany) ( 41 ), NOX1, NOX2, or NCF2 (bs-3682R, bs-3889R, bs-3891R; Bioss, Woburn, MA), ENaC-α (SPC-403D; StressMarq, Victoria, BC, Canada) followed by goat antirabbit secondary antibody conjugated to AlexaFluor 594 or AlexaFluor 488 (A11072, A27034;Thermo Fisher) and Cy3-conjugated AQP1, AlexaFluor 488-conjugated AQP2 (bs-1506R-Cy3, bs-4611R-A488; Bioss), or FITC-conjugated NKCC2 antibody (SPC-401D-FITC; StressMarq).

Techniques: Expressing, Western Blot, Isolation, Immunofluorescence, Staining

Journal: Antioxidants & Redox Signaling

Article Title: Renal NOXA1/NOX1 Signaling Regulates Epithelial Sodium Channel and Sodium Retention in Angiotensin II-induced Hypertension

doi: 10.1089/ars.2021.0047

Figure Lengend Snippet: Renal expression of ENaC is increased in male wild-type mice treated with Ang II. (A) Western blot analysis and densitometry quantification of the sodium channels NHE3, NKCC2, and NCC expression in mice treated with Ang II for 14 days. Data are fold change in protein expression adjusted for ACTB levels and relative to wild-type (mean ± SEM). (B) Western blot analysis and densitometry quantification of βENaC levels in renal lysates from wild-type and Noxa1−/− mice treated with vehicle or Ang II for 14 days. Data are protein fold change adjusted for ACTB levels and relative to control (mean ± SEM). (C) Representative immunofluorescence images and quantification of αENaC expression in the renal sections from wild-type and Noxa1−/− mice treated with vehicle or Ang II for 1 or 14 days, and stained for αENaC (red), AQP2 (green), and DAPI (blue). High magnification insets (yellow rectangle) show αENaC expression in CD epithelial cell. Scale is 100 μm. Data are fluorescence integrated density (mean ± SEM). (D) Representative immunofluorescence images and quantification of αENaC colocalization with AQP2 on the apical (A) or basolateral (BL) side of the CD cells from wild-type and Noxa1−/− mice treated with vehicle or Ang II for 1 day, and stained for αENaC (red), AQP2 (green), and DAPI (blue). Data are fluorescence integrated density (mean ± SEM). Scale is 10 μm. ACTB, β-actin; ENaC, epithelial Na+ channel; NCC, Na-Cl cotransporter; NHE3, Na-H exchanger 3.

Article Snippet: Frozen kidney sections were fixed in acetone, permeabilized in 0.1% Triton X-100, and immunofluorescent staining was performed using antibodies against NOXA1 (Ab199; a gift from Dr. Ralf Brandes, Institut für Kardiovaskuläre Physiologie, Goethe-Universität, Frankfurt am Main, Germany) ( 41 ), NOX1, NOX2, or NCF2 (bs-3682R, bs-3889R, bs-3891R; Bioss, Woburn, MA), ENaC-α (SPC-403D; StressMarq, Victoria, BC, Canada) followed by goat antirabbit secondary antibody conjugated to AlexaFluor 594 or AlexaFluor 488 (A11072, A27034;Thermo Fisher) and Cy3-conjugated AQP1, AlexaFluor 488-conjugated AQP2 (bs-1506R-Cy3, bs-4611R-A488; Bioss), or FITC-conjugated NKCC2 antibody (SPC-401D-FITC; StressMarq).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Fluorescence