anti stabilin 1 rabbit polyclonal antibody rs1  (Novus Biologicals)

Journal: Cells

Article Title: Atp1b2 Atp1b1 Knock-In Mice Exhibit a Cone–Rod Dystrophy-Like Phenotype

doi: 10.3390/cells14120878

Figure Lengend Snippet: Expression of retinoschisin in mutant and wild-type retinas. In wild-type retinas, retinoschisin (RS1; ( Aa )) and β2 ( Ab ) were co-localized ( Ac ) in the photoreceptor inner segments, outer plexiform layer, and cell bodies in the inner nuclear layer. In β2 / β1 ki retinas ( Ae – Ag ), retinoschisin was barely detectable and diffusely distributed ( Ae ). β2 was not detected in mutant retinas as expected ( Af , Ag ) is the overlay of ( Ae , Af ). As a negative control, sections were incubated with the secondary antibodies only ( Ad , Ah ). ( B ) Western blot analysis confirmed massively reduced levels of retinoschisin in mutant retinas when compared to wild-type retinas. Note that three-fold more mutant than wild-type samples were loaded. gcl: ganglion cell layer; inl: inner nuclear layer; ki: knock-in; onl: outer nuclear layer; RS1: retinoschisin; TPS: total protein stain; wt: wild-type. Scale bar: 50 µm.

Article Snippet: retinoschisin (RS1) , 1:500 (IHC) 1:500 (WB) , Proteintech Group, Inc., Rosemont, IL, USA , 24430-1-AP.

Techniques: Expressing, Mutagenesis, Negative Control, Incubation, Western Blot, Knock-In, Staining

Journal: Frontiers in Medicine

Article Title: The dose-response relationship of subretinal gene therapy with rAAV2tYF-CB-h RS1 in a mouse model of X-linked retinoschisis

doi: 10.3389/fmed.2024.1304819

Figure Lengend Snippet: Primers for Rs1 -KO genotyping.

Article Snippet: Proteins were detected by primary antibodies: polyclonal antibody against human RS1 (Atlas Antibodies, HPA059546, dilution 1:750) and β-Actin antibody monoclonal IgG sc-47778 (Santa Cruz Biotechnology, dilution 1:1000) then secondary antibodies: IRDye ® 800CW goat anti-rabbit IgG and 680RD goat anti-mouse IgG, dilutions 1:10000 (LI-COR).

Techniques: Sequencing

Journal: Frontiers in Medicine

Article Title: The dose-response relationship of subretinal gene therapy with rAAV2tYF-CB-h RS1 in a mouse model of X-linked retinoschisis

doi: 10.3389/fmed.2024.1304819

Figure Lengend Snippet: Expression cassette of the rAAV2tYF-CB-h RS1 vector. Components of rAAV2tYF-CB-h RS1 , the vector contains AAV serotype 2 inverted terminal repeats (ITRs) and an expression cassette consisting of a cytomegalovirus enhancer (CMV) and chicken β-actin promoter (CB), human RS1 complementary DNA, and a simian vacuolating virus 40 polyadenylation (SV40 PolyA) sequence and is packaged in an AAV2 capsid containing tyrosine-to-phenylalanine mutations in 3 surface-exposed tyrosine residues (Y444F, Y500F, Y730F) in the VP3 protein capsid (rAAV2tYF).

Article Snippet: Proteins were detected by primary antibodies: polyclonal antibody against human RS1 (Atlas Antibodies, HPA059546, dilution 1:750) and β-Actin antibody monoclonal IgG sc-47778 (Santa Cruz Biotechnology, dilution 1:1000) then secondary antibodies: IRDye ® 800CW goat anti-rabbit IgG and 680RD goat anti-mouse IgG, dilutions 1:10000 (LI-COR).

Techniques: Expressing, Plasmid Preparation, Virus, Sequencing

Journal: Frontiers in Medicine

Article Title: The dose-response relationship of subretinal gene therapy with rAAV2tYF-CB-h RS1 in a mouse model of X-linked retinoschisis

doi: 10.3389/fmed.2024.1304819

Figure Lengend Snippet: Characterization of retinal phenotypes in retinoschisin-1 knockout mice: rod and cone photoreceptors functional loss and cyst formation. (A) OCT images of a Rs1- KO mouse (1) and a wild-type (WT) control littermate (2) are shown at 1 month of age. The outer nuclear layer (ONL) thicknesses are indicated by a white solid bar, and cysts are indicated by white stars on the OCT image. (B) Quantification of ONL thicknesses in Rs1 -KO and WT or heterozygous control mice at 1 month of age. Rs1 -KO mice have thinner ONLs. (C) Quantification of the cyst severity in Rs1 -KO and WT or heterozygous control mice. Cyst severity score is modified from Bush et al. where (1) no cavities; (2) <30 μm; (3) 30–49 μm; (4) 50–69 μm; (5) 70–99 μm; (6) ≥100 μm. Rs1 -KO mice have cysts whereas WT or heterozygous do not. (D) (1) Waveforms in dark-adapted (DA) standard combined response (SCR) ERG using 3.0 cd·s/m 2 stimuli in Rs1 -KO and WT or heterozygous control mice. (2) b/a wave ratio is reduced in Rs1 -KO compared to WT or heterozygous controls. (E) At 1 month of age, Rs1 -KO mice have greater a-wave amplitudes compared to WT or heterozygous control mice after 0.01 cd·s/m 2 stimuli in DA conditions. Representative waveforms are shown in (1), and the statistical comparison of their a-wave amplitudes is shown in (2). (F) Cone function on ERG measured after light adaptation using 3.0 flash (1) and 5 Hz flicker (2). Amplitudes are reduced in Rs1 -KO compared to WT or heterozygous control mice. MO: month old, Rs1 -KO: retinoschisin-1 knockout, WT: wild-type or heterozygous mice, μV: Microvolt, * p < 0.05, ** p < 0.01, and **** p < 0.0001. Scale bar, 100 μm.

Article Snippet: Proteins were detected by primary antibodies: polyclonal antibody against human RS1 (Atlas Antibodies, HPA059546, dilution 1:750) and β-Actin antibody monoclonal IgG sc-47778 (Santa Cruz Biotechnology, dilution 1:1000) then secondary antibodies: IRDye ® 800CW goat anti-rabbit IgG and 680RD goat anti-mouse IgG, dilutions 1:10000 (LI-COR).

Techniques: Knock-Out, Functional Assay, Control, Modification, Comparison

Journal: Frontiers in Medicine

Article Title: The dose-response relationship of subretinal gene therapy with rAAV2tYF-CB-h RS1 in a mouse model of X-linked retinoschisis

doi: 10.3389/fmed.2024.1304819

Figure Lengend Snippet: Expression of human RS1 protein in treated retinas of Rs1 -KO mice reveals a dose-dependent response. (A) Immunoblot analysis of eyes treated with 8E9 vg, 8E8 vg, 8E7 vg doses of the AAV2tYF vector, and diluent-treated samples (Dil) retinal protein extracts. Bleb qualities were excellent for all treatment groups except for one retina treated with 8E7 dose that had a very good bleb (arrowhead). Completely untreated Rs1 -KO and WT retinas served as negative and positive controls. Each lane represents an individual eye, with three eyes analyzed per dose, 2 eyes for sham treatment, 2 eyes for WT, and 1 eye for untreated KO. Eighteen μg of protein were loaded per lane. The expected molecular weights for proteins are indicated on the left, and the antibodies used to visualize the proteins are indicated on the right. (B) RS1 expression levels relative to completely WT eyes. Quantification involved normalizing the RS1 band intensity to the actin band intensity for each sample, with the expression level presented as a percentage of WT retinal samples which is set to 100%. The results illustrate a dose-dependent increase in RS1 protein expression in Rs1 -KO mouse retinas treated with 8E7, 8E8, and 8E9 vg of rAAV2-tYF-CB-h RS1 .

Article Snippet: Proteins were detected by primary antibodies: polyclonal antibody against human RS1 (Atlas Antibodies, HPA059546, dilution 1:750) and β-Actin antibody monoclonal IgG sc-47778 (Santa Cruz Biotechnology, dilution 1:1000) then secondary antibodies: IRDye ® 800CW goat anti-rabbit IgG and 680RD goat anti-mouse IgG, dilutions 1:10000 (LI-COR).

Techniques: Expressing, Western Blot, Plasmid Preparation

Journal: Frontiers in Medicine

Article Title: The dose-response relationship of subretinal gene therapy with rAAV2tYF-CB-h RS1 in a mouse model of X-linked retinoschisis

doi: 10.3389/fmed.2024.1304819

Figure Lengend Snippet: rAAV2tYF subretinal gene therapy improves and sustains cone photoreceptor function in Rs1 -KO mice treated with the medium dose of 8E8 vg/eye over the course of 12 months. (A) Cone-dependent retinal function, assessed through light-adapted (LA) electroretinography after subjecting the eyes to a 3.0 cd·s/m 2 bright flash. Eyes treated with the 8E8 vg dose consistently displayed significantly greater b-wave amplitudes compared to sham-treated eyes and untreated eyes over 1 year. Eyes treated with 8E9 showed significance compared to the sham-treated eyes only at the experimental endpoint. (B) Representative LA waveforms were shown for eyes treated with different doses, as well as for sham-treated, and untreated eyes at 3 MPI and 7 MPI, respectively. (C) Another metric used to evaluate cone-dependent retinal function was the 5 Hz flicker test, which consistently showed that 8E8 vg dose-treated eyes outperformed sham-treated and untreated eyes throughout the treatment duration. Neither 8E9 nor 8E7 vg doses showed significance compared to the sham-treated eyes over the treatment course. (D) When subjected to a 5 Hz flicker, cones in treated eyes elicited higher amplitudes at 3 MPI, which was not observed in the sham-treated eyes or the untreated contralateral eyes. Over the course of 7 months, treated eyes retained significantly better amplitudes in cone ERGs. (E) Comparison of ERG flicker amplitudes between mice that received the medium 8E8 vg dose and WT or heterozygous control mice. At 1 MPI (2 MO), treatment with the 8E8 vg dose restored ERGs to near-WT amplitudes. (F) Waveforms from an eye that received the medium 8E8 dose and a WT eye. MO, months old, WT: wild-type or heterozygous mice, µV: Microvolt, * p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.0001.

Article Snippet: Proteins were detected by primary antibodies: polyclonal antibody against human RS1 (Atlas Antibodies, HPA059546, dilution 1:750) and β-Actin antibody monoclonal IgG sc-47778 (Santa Cruz Biotechnology, dilution 1:1000) then secondary antibodies: IRDye ® 800CW goat anti-rabbit IgG and 680RD goat anti-mouse IgG, dilutions 1:10000 (LI-COR).

Techniques: Comparison, Control

Journal: Frontiers in Medicine

Article Title: The dose-response relationship of subretinal gene therapy with rAAV2tYF-CB-h RS1 in a mouse model of X-linked retinoschisis

doi: 10.3389/fmed.2024.1304819

Figure Lengend Snippet: Subretinal gene therapy of Rs1 -KO delays loss of retinal function in standard combined response ERG. (A) Combined rod-cone function was measured by 3.0 cd·s/m 2 bright flash under dark-adapted conditions, at 1, 2, 3, 5, 7 and 12 MPI. The 8E8 vg dose treated eyes had higher b-wave amplitudes compared to diluent-injected eyes and untreated contralateral eyes. (B,C) Representative waveforms of the 3.0 cd·s/m 2 bright flash SCR ERG test are shown for all vector doses, diluent-injected eyes, and untreated eyes at 3 MPI and 7 MPI, respectively. The 8E8 dose had higher amplitudes at 3 MPI and at 7MPI. Waveforms from the same mice were used at each time point. (D) An elevated b/a ratio was observed in the 8E8 vg dose-treated eyes until 2 MPI time point and in 8E9 vg dose treated eyes until 5 MPI. (E) SCR a-wave amplitudes of eyes treated with different doses of rAAV2tYF gene therapy vector, sham-treated and untreated control mice. (F) Rod and cone function comparison between the medium 8E8 vg dose and WT or heterozygous control mice at 2 and 6 MO. MO, months old, WT: wild-type or heterozygous mice, µV: Microvolt, * p < 0.05, ** p < 0.01, and **** p < 0.0001.

Article Snippet: Proteins were detected by primary antibodies: polyclonal antibody against human RS1 (Atlas Antibodies, HPA059546, dilution 1:750) and β-Actin antibody monoclonal IgG sc-47778 (Santa Cruz Biotechnology, dilution 1:1000) then secondary antibodies: IRDye ® 800CW goat anti-rabbit IgG and 680RD goat anti-mouse IgG, dilutions 1:10000 (LI-COR).

Techniques: Injection, Plasmid Preparation, Control, Comparison

Journal: Frontiers in Medicine

Article Title: The dose-response relationship of subretinal gene therapy with rAAV2tYF-CB-h RS1 in a mouse model of X-linked retinoschisis

doi: 10.3389/fmed.2024.1304819

Figure Lengend Snippet: Subretinal gene therapy of Rs1 -KO with a dose of 8E8 vg/eye delays loss of rod photoreceptor function. (A) Rod function was measured by 0.01 cd·s/m 2 dim flash after DA. Notably, at 1, 2, and 3 MPI, some vector-treated eyes demonstrated a normalization of the hyper-normal a-wave seen in the Rs1 -KO mouse model (as shown in ). (B) B-wave amplitudes of the 0.01 cd·s/m 2 dim flash after DA showing the significantly increased b-wave amplitudes of the 8E8 vg dose compared to the sham-treated and untreated eyes. (C,D) Waveform comparisons of the 0.01 cd·s/m 2 dim flash test were made between all vector doses, diluent-injected eyes, and untreated eyes at 3 MPI and 7 MPI, respectively. (E) Rod function comparison between the medium 8E8 vg dose and WT or heterozygous control mice (WT) at 2 and 6 MO. MO: months old, WT: wild-type or heterozygous mice, µV: Microvolt, * p < 0.05, ** p ≤ 0.005, *** p ≤ 0.0005, and **** p < 0.0001.

Article Snippet: Proteins were detected by primary antibodies: polyclonal antibody against human RS1 (Atlas Antibodies, HPA059546, dilution 1:750) and β-Actin antibody monoclonal IgG sc-47778 (Santa Cruz Biotechnology, dilution 1:1000) then secondary antibodies: IRDye ® 800CW goat anti-rabbit IgG and 680RD goat anti-mouse IgG, dilutions 1:10000 (LI-COR).

Techniques: Plasmid Preparation, Injection, Comparison, Control

Journal: Frontiers in Medicine

Article Title: The dose-response relationship of subretinal gene therapy with rAAV2tYF-CB-h RS1 in a mouse model of X-linked retinoschisis

doi: 10.3389/fmed.2024.1304819

Figure Lengend Snippet: Eyes treated with the 8E8 vg dose exhibit superior cone rescue at 14 months old compared to sham-treated and untreated eyes. (A–E) Visualization of cone outer segments using peanut agglutinin. Retinal sections collected from all vector treated eyes, diluent-treated eyes, and untreated Rs1 -KO eyes at 14 months of age were processed and stained with DAPI (blue) and PNA (red) to visualize cone outer segments. (F) Quantification of cone outer segments per 100 μm of the retina indicates that there is a significant preservation of cones in eyes treated with the 8E8 vg dose compared to eyes that received sham treatment and untreated Rs1 -KO eyes. Eyes treated with all doses of the vector, as well as diluent-treated eyes, had significantly more cones than untreated eyes. Quantification was performed by three individuals masked to treatment groups. OS, outer segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer. * p < 0.05, ** p < 0.005. Scale bar, 50 μm.

Article Snippet: Proteins were detected by primary antibodies: polyclonal antibody against human RS1 (Atlas Antibodies, HPA059546, dilution 1:750) and β-Actin antibody monoclonal IgG sc-47778 (Santa Cruz Biotechnology, dilution 1:1000) then secondary antibodies: IRDye ® 800CW goat anti-rabbit IgG and 680RD goat anti-mouse IgG, dilutions 1:10000 (LI-COR).

Techniques: Plasmid Preparation, Staining, Preserving

Journal: Frontiers in Medicine

Article Title: The dose-response relationship of subretinal gene therapy with rAAV2tYF-CB-h RS1 in a mouse model of X-linked retinoschisis

doi: 10.3389/fmed.2024.1304819

Figure Lengend Snippet: Medium dose (8E8 vg) of subretinal rAAV2tYF gene therapy shows improvement in functional vision. (A) Under normal fluorescent ceiling light, Rs1- KO mice treated with 8E8 vg dosage of the rAAV2tYF subretinal gene therapy took significantly less time to locate the platform compared to untreated Rs1- KO mice but not compared to the sham-treated mice at both time points (4–6 and 9–11 MO). (B) In dark testing conditions (dim red lighting), mice treated with the 8E8 vg dose of the gene therapy took significantly less time to locate the platform compared to untreated Rs1- KO mice at younger ages but not at older ages. No statistical difference was observed for any of the vector treated eyes compared to the sham-treated eyes. (C) Confidence intervals were employed to show the data spread. No statistically significant difference or clear trend towards improvement at older age compared to the untreated eyes was observed. MO: months old, * p < 0.05.

Article Snippet: Proteins were detected by primary antibodies: polyclonal antibody against human RS1 (Atlas Antibodies, HPA059546, dilution 1:750) and β-Actin antibody monoclonal IgG sc-47778 (Santa Cruz Biotechnology, dilution 1:1000) then secondary antibodies: IRDye ® 800CW goat anti-rabbit IgG and 680RD goat anti-mouse IgG, dilutions 1:10000 (LI-COR).

Techniques: Functional Assay, Plasmid Preparation

Journal: Emerging Microbes & Infections

Article Title: MERS-CoV seroconversion amongst Malaysian Hajj pilgrims returning from the Middle East, 2016–2018: results from the MERCURIAL multiyear prospective cohort study

doi: 10.1080/22221751.2023.2208678

Figure Lengend Snippet: Evaluation of pre- and post-Hajj IgG antibody for MERS-CoV using rS1 Enzyme-Linked Immunosorbent Assay (ELISA).

Article Snippet: Paired pre- and post-Hajj serum samples were tested for the presence of MERS-CoV-specific antibodies using the commercially available anti-MERS-CoV IgG rS1 ELISA kit (EUROIMMUN, Lübeck, Germany) according to the manufacturer’s guidelines.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Emerging Microbes & Infections

Article Title: MERS-CoV seroconversion amongst Malaysian Hajj pilgrims returning from the Middle East, 2016–2018: results from the MERCURIAL multiyear prospective cohort study

doi: 10.1080/22221751.2023.2208678

Figure Lengend Snippet: Case findings of Twelve Seroconverted Hajj Pilgrims.

Article Snippet: Paired pre- and post-Hajj serum samples were tested for the presence of MERS-CoV-specific antibodies using the commercially available anti-MERS-CoV IgG rS1 ELISA kit (EUROIMMUN, Lübeck, Germany) according to the manufacturer’s guidelines.

Techniques: Enzyme-linked Immunosorbent Assay