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