rabbit polyclonal anti-rbpms primary antibody  (PhosphoSolutions)

Journal: Scientific Reports

Article Title: Hypoxia-mediated rescue of retinal ganglion cells deficient in mitochondrial complex I is independent of the hypoxia-inducible factor pathway

doi: 10.1038/s41598-024-75916-x

Figure Lengend Snippet: Deletion of vhl does not prevent degeneration of ndufs4 -deficient retinal ganglion cells. (A) To assess for cell-specific recombination of floxed vhl , a flattened retina from a Vglut2-Cre;vhl loxP/loxP mouse was serially sectioned in 40-µm increments to collect tissue from the outermost to the innermost retinal layers (samples 1–5). PCR was performed on purified genomic DNA from each section to reveal the presence of vhl loxP/loxP that is non-recombined (2 loxP sites, 460 bp) and recombined (1 loxP site, 260 bp). The presence of the Vglut2-Cre transgene in the genomic DNA was also confirmed by PCR. The schematics to the right depict the beginning of the vhl locus pre- and post-recombination, showing the positions of the loxP sites flanking Exon 1 and the expected PCR product sizes resulting from a reaction in which two forward primers (F1 and F2) and one common reverse primer (Rcomm) are used. See Supplementary Fig. online for un-cropped gels. (B, C) Representative images of retinal flat mounts from control mice without the Cre transgene (left panel) and from mice with the indicated heterozygosity or homozygosity status for deletion of ndufs4 and vhl from RGCs at (B) P30 and (C) P45 time points. RGCs are immunolabeled with RNA-Binding Protein 1 (RBPMS1, green). Bar, 20 μm. In each graph below, the density of RGC somas for each genotype is quantified at distances of 0.5, 1.0, and 1.5 mm from the optic nerve head. Individual data points are overlaid on each bar. Statistical comparisons between groups are indicated above the bars, with the following significance designations: ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Retinas were isolated, blocked in 5% goat serum in PBS with 0.3% Triton X-100, incubated with rabbit polyclonal anti-RBPMS1 primary antibody (1:500; Novus, NBP2-20112) in block for 5 days at 4 °C, and then incubated with anti-rabbit Alexa Fluor 488 (1:500; Invitrogen) overnight at 4 °C.

Techniques: Purification, Control, Immunolabeling, RNA Binding Assay

Journal: Scientific Reports

Article Title: Hypoxia-mediated rescue of retinal ganglion cells deficient in mitochondrial complex I is independent of the hypoxia-inducible factor pathway

doi: 10.1038/s41598-024-75916-x

Figure Lengend Snippet: Retinal ganglion cell-specific deletion of Hif1α and Hif2α . (A) To confirm cell-specific recombination of floxed Hif1α and Hif2α , a flattened retina from a Vglut2-Cre; Hif1 α loxP/loxP ; Hif2 α loxP/loxP mouse was serially sectioned in 40-µm increments to collect tissue from the outermost to the innermost retinal layers (samples 1–5). PCR was performed on purified genomic DNA from each section to demonstrate the presence of Hif1 α loxP/loxP and Hif2 α loxP/loxP alleles that are non-recombined (2 loxP sites) and recombined (1 loxP site). The presence of the Vglut2-Cre transgene in the genomic DNA was also confirmed by PCR. The schematics to the right depict the beginning of the floxed Hif1α and Hif2α loci pre- and post-recombination, showing the positions of the loxP sites flanking Exon 2 of each gene. The positions of each of the two forward primers (F1 and F2) and one common reverse primer (Rcomm) used for each gene are also depicted, as well as the expected PCR product sizes for each reaction when all three primers are combined. Note that for Hif1α , the locations of the forward primers relative to each loxP site result in the post-recombination amplification product being slightly larger than for the non-recombined product. See Supplementary Fig. S2 online for un-cropped gels. (B) Retinal flat mount from a P60 Vglut2-Cre; Hif1 α loxP/loxP ; Hif2 α loxP/loxP mouse stained for RBPMS1 demonstrates healthy RGC soma morphology and density. Bar, 20 μm. (C) Electron micrograph of an optic nerve cross section from a P60 Vglut2-Cre; Hif1 α loxP/loxP ; Hif2 α loxP/loxP mouse demonstrates abundant RGC axons with normal myelination. Bar, 5 μm.

Article Snippet: Retinas were isolated, blocked in 5% goat serum in PBS with 0.3% Triton X-100, incubated with rabbit polyclonal anti-RBPMS1 primary antibody (1:500; Novus, NBP2-20112) in block for 5 days at 4 °C, and then incubated with anti-rabbit Alexa Fluor 488 (1:500; Invitrogen) overnight at 4 °C.

Techniques: Purification, Amplification, Staining

Journal: Scientific Reports

Article Title: Hypoxia-mediated rescue of retinal ganglion cells deficient in mitochondrial complex I is independent of the hypoxia-inducible factor pathway

doi: 10.1038/s41598-024-75916-x

Figure Lengend Snippet: Neuroprotection of P60 ndufs4 -deficient retinal ganglion cell somas by hypoxia is not dependent on an intact HIF pathway . Representative images of RBPMS1-labeled retinal flat mounts from P60 mice with RGC-specific deletion of both Hif1α and Hif2α. When the mice are raised under normoxia, the density of RGC somas (immunolabeled for RBPMS1) is reduced in mice that are also homozygous for deletion of ndufs4 within RGCs (middle panel) compared to those with one intact copy (left panel). With continuous exposure to 11% O 2 beginning at P25, RGCs with homozygous deletion of ndufs4 (right panel) maintain a normal cell density. Bar, 20 μm. The graph depicts RGC soma density at 0.5, 1.0, and 1.5 mm distances from the optic nerve head, with the RGC ndufs4 genotype and the ambient O 2 concentration indicated below. Individual data points are overlaid on each bar. Statistical comparisons between groups are indicated above the bars, with the following significance designations: ns, not significant; ***, p < 0.001.

Article Snippet: Retinas were isolated, blocked in 5% goat serum in PBS with 0.3% Triton X-100, incubated with rabbit polyclonal anti-RBPMS1 primary antibody (1:500; Novus, NBP2-20112) in block for 5 days at 4 °C, and then incubated with anti-rabbit Alexa Fluor 488 (1:500; Invitrogen) overnight at 4 °C.

Techniques: Labeling, Immunolabeling, Concentration Assay

Journal: Scientific Reports

Article Title: Hypoxia-mediated rescue of retinal ganglion cells deficient in mitochondrial complex I is independent of the hypoxia-inducible factor pathway

doi: 10.1038/s41598-024-75916-x

Figure Lengend Snippet: The partial rescue of ndufs4 - deficient retinal ganglion cells by hypoxia at P90 is maintained in the absence of an intact HIF pathway . (A, B) Representative images of RBPMS1-labeled retinal flat mounts (A) or optic nerve cross sections (B) from P90 mice with RGC-specific homozygous deletion of both Hif1α and Hif2α. The left panels depict tissue in which there is also heterozygous deletion of ndufs4 from RGCs, while in the right panels there is homozygous deletion of ndufs4 from RGCs. From P25 to P90, the mice were raised under 21% O 2 (top panels) or 11% O 2 (bottom panels). Bar, 20 μm. The graphs to the right quantify RGC soma densities at three distances from the optic nerve head (A) and RGC axon densities across entire optic nerve cross sections (B). Individual data points are overlaid on each bar. Statistical comparisons between groups are indicated above the bars, with the following significance designations: ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. (C) Electron micrographs of optic nerve cross sections from P90 Vglut2-Cre; ndufs4 loxP/loxP ; Hif1 α loxP/loxP ; Hif2 α loxP/loxP mice raised under normoxia (top panels) or hypoxia (bottom panels). Continuous hypoxia reduced RGC axon loss and surrounding fibrosis. The thickening and duplication of myelin sheaths on surviving axons (higher magnification images) were less common in hypoxia-treated mice, but scattered examples were still observed. Black bars, 10 μm. White bars, 0.5 μm.

Article Snippet: Retinas were isolated, blocked in 5% goat serum in PBS with 0.3% Triton X-100, incubated with rabbit polyclonal anti-RBPMS1 primary antibody (1:500; Novus, NBP2-20112) in block for 5 days at 4 °C, and then incubated with anti-rabbit Alexa Fluor 488 (1:500; Invitrogen) overnight at 4 °C.

Techniques: Labeling

Journal: International Journal of Molecular Sciences

Article Title: A Fair Assessment of Evaluation Tools for the Murine Microbead Occlusion Model of Glaucoma

doi: 10.3390/ijms22115633

Figure Lengend Snippet: Intracameral microbead injection led to a mild glaucoma phenotype. ( a ) Intraocular pressure (IOP), measured with a rebound tonometer, was increased in microbead-injected eyes at two weeks post-injection, in comparison to the contralateral eyes. Of the 10 mice, 4 did not show elevated IOP (defined as an IOP lower than the mean IOP value of the contralateral eye + one standard deviation) (shown in red). Unpaired two-tailed t -test, ** p ≤ 0.01, n = 10. ( b ) RGC density of entire RBPMS-stained flatmounts was calculated by an automated deep learning tool (RGCode) and revealed a mild RGC loss at five weeks post-injection, as compared to contralateral eyes. Unpaired two-tailed t -test, *** p ≤ 0.001, n = 10. ( c ) Representative photomicrographs of mid-peripheral regions of RBPMS-stained flatmounts revealed no evidently visible changes in RGC densities across contralateral control and microbead-injected eyes. Scale bar = 50 µm. CL = contralateral eyes and MB = microbead-injected eyes.

Article Snippet: Following a 15 min freezing step in PBS with 0.5% Triton X-100 (VWR) at −80 °C, the flatmounts were incubated overnight with primary rabbit anti-RBPMS antibody (1/250, PhosphoSolutions, Aurora, CO, USA) in PBS with 2% Triton X-100 and 2% pre-immune donkey serum at room temperature.

Techniques: Injection, Comparison, Standard Deviation, Two Tailed Test, Staining, Control

Journal: Translational Vision Science & Technology

Article Title: Software for Quantifying and Batch Processing Images of Brn3a and RBPMS Immunolabelled Retinal Ganglion Cells in Retinal Wholemounts

doi: 10.1167/tvst.9.6.28

Figure Lengend Snippet: Representative immunolabeled images for Brn3a and RBPMS (A–E) and the corresponding processed images ready for automated counting (F–J). Scale bar = 25 µm, 10 × magnification immunolabeled confocal photomicrographs, 150 × 150 µm cropped frames. Arrows demonstrate faintly visible cells in the background with poor border definition, which were excluded from both manual and automated counts.

Article Snippet: Retinas were permeabilized with phosphate buffered saline (PBS; 137 mM NaCl, 5.4 mM KCl, 1.28 mM NaH 2 PO 4 , 7 mM Na 2 HPO 4 ; and pH 7.4) containing 1% Triton X-100 (PBST-1%), blocked in PBST-1% containing 3% (v/v) normal horse serum, then incubated for three days at 4°C in the same solution containing either goat anti-Brn3a primary antibody (1:600; SC-31984; Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-RBPMS primary antibody (1:500; ABN1362; Merck Millipore, Bayswater, Victoria, Australia).

Techniques: Immunolabeling

Journal: Translational Vision Science & Technology

Article Title: Software for Quantifying and Batch Processing Images of Brn3a and RBPMS Immunolabelled Retinal Ganglion Cells in Retinal Wholemounts

doi: 10.1167/tvst.9.6.28

Figure Lengend Snippet: Examples of counting rules followed by manual observers (please note zoomed-in images are not to scale). (A) Brn3a immunolabelled RGCs. (B) RBPMS immunolabelled RGCs. Arrows (→) highlight faintly visible cells with poor border definition and dashed arrows; (–>) demarcate cells of which <50% of the cell was visualized on the image border, both of which were excluded from manual counts.

Article Snippet: Retinas were permeabilized with phosphate buffered saline (PBS; 137 mM NaCl, 5.4 mM KCl, 1.28 mM NaH 2 PO 4 , 7 mM Na 2 HPO 4 ; and pH 7.4) containing 1% Triton X-100 (PBST-1%), blocked in PBST-1% containing 3% (v/v) normal horse serum, then incubated for three days at 4°C in the same solution containing either goat anti-Brn3a primary antibody (1:600; SC-31984; Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-RBPMS primary antibody (1:500; ABN1362; Merck Millipore, Bayswater, Victoria, Australia).

Techniques:

Journal: Translational Vision Science & Technology

Article Title: Software for Quantifying and Batch Processing Images of Brn3a and RBPMS Immunolabelled Retinal Ganglion Cells in Retinal Wholemounts

doi: 10.1167/tvst.9.6.28

Figure Lengend Snippet: ICC (95% CI) of Immunohistochemical Labels (Correlation Between all Three Observers)

Article Snippet: Retinas were permeabilized with phosphate buffered saline (PBS; 137 mM NaCl, 5.4 mM KCl, 1.28 mM NaH 2 PO 4 , 7 mM Na 2 HPO 4 ; and pH 7.4) containing 1% Triton X-100 (PBST-1%), blocked in PBST-1% containing 3% (v/v) normal horse serum, then incubated for three days at 4°C in the same solution containing either goat anti-Brn3a primary antibody (1:600; SC-31984; Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-RBPMS primary antibody (1:500; ABN1362; Merck Millipore, Bayswater, Victoria, Australia).

Techniques: Immunohistochemical staining

Journal: Translational Vision Science & Technology

Article Title: Software for Quantifying and Batch Processing Images of Brn3a and RBPMS Immunolabelled Retinal Ganglion Cells in Retinal Wholemounts

doi: 10.1167/tvst.9.6.28

Figure Lengend Snippet: Bland-Altman Tests - Bias (95% Limits of Agreement) (GT versus Automated Counts)

Article Snippet: Retinas were permeabilized with phosphate buffered saline (PBS; 137 mM NaCl, 5.4 mM KCl, 1.28 mM NaH 2 PO 4 , 7 mM Na 2 HPO 4 ; and pH 7.4) containing 1% Triton X-100 (PBST-1%), blocked in PBST-1% containing 3% (v/v) normal horse serum, then incubated for three days at 4°C in the same solution containing either goat anti-Brn3a primary antibody (1:600; SC-31984; Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-RBPMS primary antibody (1:500; ABN1362; Merck Millipore, Bayswater, Victoria, Australia).

Techniques:

Journal: Translational Vision Science & Technology

Article Title: Software for Quantifying and Batch Processing Images of Brn3a and RBPMS Immunolabelled Retinal Ganglion Cells in Retinal Wholemounts

doi: 10.1167/tvst.9.6.28

Figure Lengend Snippet: Bland-Altman Plots of Ground Truth (GT) versus Automated Counts in both naive and injured retina. The uninterrupted line (___) indicates the bias. The dashed lines (—) indicate the 95% limits of agreement. Group 1, Brn3a OHT model ( n = 80 frames for both naïve and injured retinas); group 2, RBPMS naïve cohort ( n = 48 frames for naïve retinas only); group 3, RBPMS NMDA model ( n = 48 frames for both naïve and injured retinas).

Article Snippet: Retinas were permeabilized with phosphate buffered saline (PBS; 137 mM NaCl, 5.4 mM KCl, 1.28 mM NaH 2 PO 4 , 7 mM Na 2 HPO 4 ; and pH 7.4) containing 1% Triton X-100 (PBST-1%), blocked in PBST-1% containing 3% (v/v) normal horse serum, then incubated for three days at 4°C in the same solution containing either goat anti-Brn3a primary antibody (1:600; SC-31984; Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-RBPMS primary antibody (1:500; ABN1362; Merck Millipore, Bayswater, Victoria, Australia).

Techniques:

Journal: Translational Vision Science & Technology

Article Title: Software for Quantifying and Batch Processing Images of Brn3a and RBPMS Immunolabelled Retinal Ganglion Cells in Retinal Wholemounts

doi: 10.1167/tvst.9.6.28

Figure Lengend Snippet: Linear Regression Analysis – Slope of Best Fit (R2) (GT vs Automated Counts)

Article Snippet: Retinas were permeabilized with phosphate buffered saline (PBS; 137 mM NaCl, 5.4 mM KCl, 1.28 mM NaH 2 PO 4 , 7 mM Na 2 HPO 4 ; and pH 7.4) containing 1% Triton X-100 (PBST-1%), blocked in PBST-1% containing 3% (v/v) normal horse serum, then incubated for three days at 4°C in the same solution containing either goat anti-Brn3a primary antibody (1:600; SC-31984; Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-RBPMS primary antibody (1:500; ABN1362; Merck Millipore, Bayswater, Victoria, Australia).

Techniques:

Journal: Translational Vision Science & Technology

Article Title: Software for Quantifying and Batch Processing Images of Brn3a and RBPMS Immunolabelled Retinal Ganglion Cells in Retinal Wholemounts

doi: 10.1167/tvst.9.6.28

Figure Lengend Snippet: Linear regression analysis in naïve and injured retina demonstrated a strong linear correlation between the averaged manual counts of three observers (Ground Truth [GT]) and automated cell counts. Group 1, Brn3a OHT model ( n = 80 frames for both naïve and injured retinas); group 2, RBPMS naïve cohort ( n = 48 frames for naïve retinas only); group 3, RBPMS NMDA model ( n = 48 frames for both naïve and injured retinas).

Article Snippet: Retinas were permeabilized with phosphate buffered saline (PBS; 137 mM NaCl, 5.4 mM KCl, 1.28 mM NaH 2 PO 4 , 7 mM Na 2 HPO 4 ; and pH 7.4) containing 1% Triton X-100 (PBST-1%), blocked in PBST-1% containing 3% (v/v) normal horse serum, then incubated for three days at 4°C in the same solution containing either goat anti-Brn3a primary antibody (1:600; SC-31984; Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-RBPMS primary antibody (1:500; ABN1362; Merck Millipore, Bayswater, Victoria, Australia).

Techniques: