Transfection:Article Title: ESCO2 Interacts with TRF1/2 and Facilitates Telomere Maintenance
Article Snippet: .. Briefly, cells harvested at 72 h after siRNA transfection were fixed for 10 min with 4% paraformaldehyde and then permeabilized for 15 min in 0.5% Triton X-100 followed by blocking with 3% bovine serum albumin for 1 h. Cells were then incubated with 53BP1 antibody (NB100-904, Novus) for 2 h at 37 °C and a secondary antibody for 1 h at room temperature. .. For FISH, an additional incubation step with an FITC-TelC-PNA probe (Panagene, Daejeon, Republic of Korea) was performed after secondary antibody incubation at 37 °C for 2 h. Cells were then washed twice with wash buffer 1 (10 mM Tris-HCl [pH 7.4], 70% formamide) and three times with wash buffer 2 (100 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20).
Article Title: ESCO2 Interacts with TRF1/2 and Facilitates Telomere Maintenance
Article Snippet: .. Briefly, cells harvested at 72 h after siRNA transfection were fixed for 10 min with 4% paraformaldehyde and then permeabilized for 15 min in 0.5% Triton X-100 followed by blocking with 3% bovine serum albumin for 1 h. Cells were then incubated with 53BP1 antibody (NB100-904, Novus) for 2 h at 37 ◦C and a secondary antibody for 1 h at room temperature. .. For FISH, an additional incubation step with an FITC-TelC-PNA probe (Panagene, Daejeon, Republic of Korea) was performed after secondary antibody incubation at 37 ◦C for 2 h. Cells were then washed twice with wash buffer 1 (10 mM Tris-HCl [pH 7.4], 70% formamide) and three times with wash buffer 2 (100 mM TrisHCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20).
Blocking Assay:Article Title: ESCO2 Interacts with TRF1/2 and Facilitates Telomere Maintenance
Article Snippet: .. Briefly, cells harvested at 72 h after siRNA transfection were fixed for 10 min with 4% paraformaldehyde and then permeabilized for 15 min in 0.5% Triton X-100 followed by blocking with 3% bovine serum albumin for 1 h. Cells were then incubated with 53BP1 antibody (NB100-904, Novus) for 2 h at 37 °C and a secondary antibody for 1 h at room temperature. .. For FISH, an additional incubation step with an FITC-TelC-PNA probe (Panagene, Daejeon, Republic of Korea) was performed after secondary antibody incubation at 37 °C for 2 h. Cells were then washed twice with wash buffer 1 (10 mM Tris-HCl [pH 7.4], 70% formamide) and three times with wash buffer 2 (100 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20).
Article Title: ESCO2 Interacts with TRF1/2 and Facilitates Telomere Maintenance
Article Snippet: .. Briefly, cells harvested at 72 h after siRNA transfection were fixed for 10 min with 4% paraformaldehyde and then permeabilized for 15 min in 0.5% Triton X-100 followed by blocking with 3% bovine serum albumin for 1 h. Cells were then incubated with 53BP1 antibody (NB100-904, Novus) for 2 h at 37 ◦C and a secondary antibody for 1 h at room temperature. .. For FISH, an additional incubation step with an FITC-TelC-PNA probe (Panagene, Daejeon, Republic of Korea) was performed after secondary antibody incubation at 37 ◦C for 2 h. Cells were then washed twice with wash buffer 1 (10 mM Tris-HCl [pH 7.4], 70% formamide) and three times with wash buffer 2 (100 mM TrisHCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20).
Article Title: Hyperactivation of distinct thalamic nuclei differentially impairs sleep physiology in rats.
Article Snippet: Brains were sliced (50 μm thick coronal sections) using a vibratome (VT1000 S, Leica Microsystems, Wetzlar, Germany). .. Free-floating sections were washed in PBS and blocked with 1% bovine serum albumin in PBS containing 0.25% Triton X-100 (PBS-T) at room temperature for 2 h. Afterwards, sections were incubated with Chicken anti-mCherry antibody (1:1000, Biotechne NBP2-25158, Novus Biologicals) in a blocking solution for 24 h at 4 °C followed by three washes in PBS. .. Then, the slices were incubated with Cy3 conjugated donkey anti-chicken secondary antibody (1:500, Jackson Immunoresearch 703-165-155) for 2 h at room temperature.
Incubation:Article Title: ESCO2 Interacts with TRF1/2 and Facilitates Telomere Maintenance
Article Snippet: .. Briefly, cells harvested at 72 h after siRNA transfection were fixed for 10 min with 4% paraformaldehyde and then permeabilized for 15 min in 0.5% Triton X-100 followed by blocking with 3% bovine serum albumin for 1 h. Cells were then incubated with 53BP1 antibody (NB100-904, Novus) for 2 h at 37 °C and a secondary antibody for 1 h at room temperature. .. For FISH, an additional incubation step with an FITC-TelC-PNA probe (Panagene, Daejeon, Republic of Korea) was performed after secondary antibody incubation at 37 °C for 2 h. Cells were then washed twice with wash buffer 1 (10 mM Tris-HCl [pH 7.4], 70% formamide) and three times with wash buffer 2 (100 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20).
Article Title: Norrin Ameliorates Retinal Ganglion Cell Apoptosis by Normalizing VEGF and PEDF Dysregulation in Diabetic Retinopathy.
Article Snippet: .. The retinas were permeabilized with 1.0% Triton X-100, blocked with 2% bovine serum albumin in Tris-buffered saline plus Tween, and incubated with a monoclonal antibody against norrin (1:200; Novus Biologicals). .. The retinas were then sequentially incubated with an FITC-conjugated goat anti-rabbit IgG (1:200; Invitrogen) for 2 h and an Alexa FluorTM 647-conjugated GFAP monoclonal antibody (1:200; Invitrogen) for 6 h. Norrin and GFAP expression patterns were co-visualized by confocal microscopy, and norrin expression was quantitatively analyzed by measuring fluorescence intensity, expressed as relative fluorescence intensity normalized to normal control mice (n = 6 retinas).
Article Title: Glycosylation-driven necroptosis in retinal degeneration: dual rescue by AAV8 gene therapy and RIPK1 inhibition.
Article Snippet: Eyes were dehydrated in 30% sucrose (#107651, Merck, US) and embedded in optimal cutting temperature (OCT) compound (#4583, Sakura, US) at -80 °C. .. Retinal sections were prepared (10 μm) from the frozen tissue and were washed in PBS and blocked with 1% bovine serum albumin (BSA) containing 1% normal goat serum for 1 h. The sections were incubated with primary antibodies against GFAP (1:200, ab7260, Abcam, US), RIP3 (1:100, NBP1-77299, Novus, US), enolase-1 (1:1000, ab155102, Abcam, US), S-arrestin (1:1000, NBP2-25161, Novus, US), and LC3B (1:100, ab51520, Abcam, US). .. The sections were then incubated with the corresponding Alexa Fluor-conjugated secondary antibodies (1:100, Invitrogen, US), and counterstaining was performed using DAPI (1:500, #10236276001, Sigma, US).
Article Title: Norrin Ameliorates Retinal Ganglion Cell Apoptosis by Normalizing VEGF and PEDF Dysregulation in Diabetic Retinopathy
Article Snippet: .. The retinas were permeabilized with 1.0% Triton X-100, blocked with 2% bovine serum albumin in Tris-buffered saline plus Tween, and incubated with a monoclonal antibody against norrin (1:200; Novus Biologicals). .. The retinas were then sequentially incubated with an FITC-conjugated goat anti-rabbit IgG (1:200; Invitrogen) for 2 h and an Alexa FluorTM 647-conjugated GFAP monoclonal antibody (1:200; Invitrogen) for 6 h. Norrin and GFAP expression patterns were co-visualized by confocal microscopy, and norrin expression was quantitatively analyzed by measuring fluorescence intensity, expressed as relative fluorescence intensity normalized to normal control mice ( n = 6 retinas).
Article Title: ESCO2 Interacts with TRF1/2 and Facilitates Telomere Maintenance
Article Snippet: .. Briefly, cells harvested at 72 h after siRNA transfection were fixed for 10 min with 4% paraformaldehyde and then permeabilized for 15 min in 0.5% Triton X-100 followed by blocking with 3% bovine serum albumin for 1 h. Cells were then incubated with 53BP1 antibody (NB100-904, Novus) for 2 h at 37 ◦C and a secondary antibody for 1 h at room temperature. .. For FISH, an additional incubation step with an FITC-TelC-PNA probe (Panagene, Daejeon, Republic of Korea) was performed after secondary antibody incubation at 37 ◦C for 2 h. Cells were then washed twice with wash buffer 1 (10 mM Tris-HCl [pH 7.4], 70% formamide) and three times with wash buffer 2 (100 mM TrisHCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20).
Article Title: Hyperactivation of distinct thalamic nuclei differentially impairs sleep physiology in rats.
Article Snippet: Brains were sliced (50 μm thick coronal sections) using a vibratome (VT1000 S, Leica Microsystems, Wetzlar, Germany). .. Free-floating sections were washed in PBS and blocked with 1% bovine serum albumin in PBS containing 0.25% Triton X-100 (PBS-T) at room temperature for 2 h. Afterwards, sections were incubated with Chicken anti-mCherry antibody (1:1000, Biotechne NBP2-25158, Novus Biologicals) in a blocking solution for 24 h at 4 °C followed by three washes in PBS. .. Then, the slices were incubated with Cy3 conjugated donkey anti-chicken secondary antibody (1:500, Jackson Immunoresearch 703-165-155) for 2 h at room temperature.
Article Title: TMEM106B mediates ACE2-independent replication of the SARS-CoV-2 S-E484D variant in airway-derived cell models
Article Snippet: .. After permeabilization, cells were incubated in 1% bovine serum albumin (BSA) and 10% FBS in PBS containing 0.1% Tween-20 (PBST) at room temperature for 1 h. Samples were then incubated in primary mouse monoclonal anti-HA (Biolegend, clone 16B12, 1:500) and rabbit polyclonal anti Rab7a (Novus Biologicals #NBP1-87174, 1:500) antibodies at 4 °C overnight. .. After washing with 1X HBBS, samples were incubated in a goat anti-mouse secondary antibody conjugated to Alexa Fluor Plus 488 (Invitrogen, Cat# A-11029, 1:1000) and goat anti-rabbit secondary antibody conjugated to Alexa Fluor Plus 568 (Invitrogen, Cat# A-11036, 1:1000) at room temperature for 1 h. Nuclei were stained with DAPI diluted in 1X HBBS at room temperature for 10 min.
Saline:Article Title: Norrin Ameliorates Retinal Ganglion Cell Apoptosis by Normalizing VEGF and PEDF Dysregulation in Diabetic Retinopathy.
Article Snippet: .. The retinas were permeabilized with 1.0% Triton X-100, blocked with 2% bovine serum albumin in Tris-buffered saline plus Tween, and incubated with a monoclonal antibody against norrin (1:200; Novus Biologicals). .. The retinas were then sequentially incubated with an FITC-conjugated goat anti-rabbit IgG (1:200; Invitrogen) for 2 h and an Alexa FluorTM 647-conjugated GFAP monoclonal antibody (1:200; Invitrogen) for 6 h. Norrin and GFAP expression patterns were co-visualized by confocal microscopy, and norrin expression was quantitatively analyzed by measuring fluorescence intensity, expressed as relative fluorescence intensity normalized to normal control mice (n = 6 retinas).
Article Title: Norrin Ameliorates Retinal Ganglion Cell Apoptosis by Normalizing VEGF and PEDF Dysregulation in Diabetic Retinopathy
Article Snippet: .. The retinas were permeabilized with 1.0% Triton X-100, blocked with 2% bovine serum albumin in Tris-buffered saline plus Tween, and incubated with a monoclonal antibody against norrin (1:200; Novus Biologicals). .. The retinas were then sequentially incubated with an FITC-conjugated goat anti-rabbit IgG (1:200; Invitrogen) for 2 h and an Alexa FluorTM 647-conjugated GFAP monoclonal antibody (1:200; Invitrogen) for 6 h. Norrin and GFAP expression patterns were co-visualized by confocal microscopy, and norrin expression was quantitatively analyzed by measuring fluorescence intensity, expressed as relative fluorescence intensity normalized to normal control mice ( n = 6 retinas).
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