After transcardial perfusion with saline (0.9% NaCl diluted in ddH2O) followed by 4% paraformaldehyde (PFA), the eyes, optic nerves and brain were harvested and post-fixed in 4% PFA for 24 hours. The eyes were then transferred to phosphate buffered saline (PBS) and the optic nerve and brain placed in 30% sucrose for cryoprotection. The optic nerve was sectioned using a sledge microtome, cutting longitudinally at 15 μm. The brain was sectioned coronally at 30 μm. All tissue sections were included for analysis. Retinas, optic nerves, and brain tissue were kept at 4° Celsius overnight using the following antibodies: rabbit-anti-RBPMS (PhosphoSolutions, 1:1000), to label RGCs; rabbit- or guinea pig-anti-GFP (SySy; 1:1000), to enhance GFP signal; rabbit-anti-p-S6 (Cell Signaling Technology; 1:250), to label phosphorylated S6 protein; mouse anti-SMI-32 (Sternberger monoclonals, 1:2000) to label alpha and other large soma RGC types; rabbit anti-melanopsin (Advanced Targeting Systems, 1:1000). For secondary detection, Alexa Fluor 488 goat anti-rabbit or anti-guinea pig (1:1000; Life Technologies), or Alexa Fluor 594 goat anti-rabbit (1:1000; Life Technologies) were used. Immunostained tissues were imaged with with an epifluorescence microscope (Zeiss Axio imager 2 with HR Zeiss camera, 10X and 20X objectives).
Rabbit anti rbpms
Manufactured by PhosphoSolutions
Rabbit anti-RBPMS is a primary antibody that recognizes the RNA-binding protein (RBPMS) in rabbit samples. RBPMS is involved in RNA metabolism and cellular processes. This antibody can be used for research purposes to detect and study the expression of RBPMS in various biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 594 goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Rabbit anti ps6, by Cell Signaling Technology (2 mentions)
Alexa fluor 488 goat anti rabbit or anti guinea pig, by Thermo Fisher Scientific (2 mentions)
Donkey anti rabbit 488, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 488 donkey anti chicken, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 488 donkey anti rabbit, by Jackson ImmunoResearch (1 mentions)
Rabbit anti βgal, by Rockland Immunochemicals (1 mentions)
Goat anti brn3, by Santa Cruz Biotechnology (1 mentions)
Goat anti chat, by Merck Group (1 mentions)
Donkey anti rabbit cy3, by Jackson ImmunoResearch (1 mentions)
Donkey anti goat cy3, by Jackson ImmunoResearch (1 mentions)
Cv1200 confocal microscope, by Olympus (1 mentions)
Rabbit anti trpv4, by LifeSpan BioSciences (1 mentions)
Mouse anti gfp, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti calretinin, by Swant (1 mentions)
Rabbit anti calbindin, by Swant (1 mentions)
Mouse anti gad67, by Merck Group (1 mentions)
Mouse anti brn3a, by Merck Group (1 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (1 mentions)
Rabbit anti gfap, by Agilent Technologies (1 mentions)
8 protocols using rabbit anti rbpms
1
Immunostaining and Imaging Protocol for Retinal, Optic Nerve, and Brain Tissues
2
Visualizing Retinal Cell Types in Mice
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SertCre/+ TaumGFP-NLS-LacZ P7 mice were euthanized with an overdose of pentobarbital-xylazine and perfused transcardially with 4% PFA in 0.12 M PB. Eyes were postfixed overnight in the same fixative, cryoprotected with 10% sucrose in PB during 2 days. Eyes were then embedded with 7.5% gelatin and 10% sucrose in PB, frozen 1 minute in isopentane at −55 °C, sectioned with a cryostat (20 µm; Leica CM 3000) and collected on superfrost slides. Slides were stored at −80 °C.
Sections were blocked with PGTx (0.2% gelatin and 0.25% Triton X-100 in PBS). Antibodies were diluted in PGTx: Rabbit anti-βGal (1/5000, Rockland), Goat anti-ChAT (1/200, Chemicon), Donkey anti-rabbit 488 and Donkey anti-goat Cy3 (1/500; Jackson Immunoresearch).
For retinal whole-mount immunostaining, retinas were first permeabilized in 1% Triton X-100 for 30 minutes, blocked in PHTx (0.1% Triton X-100 10% horse serum in PBS). Antibodies were diluted in PHTx: Rabbit anti-βGal (1/5000, Rockland), Goat anti-Brn3 (1/200, C-13, Santa Cruz), Rabbit anti-RBPMS (1/500, Phosphosolutions, # 1830), Donkey anti-rabbit AlexaFluor488, Donkey anti-chicken AlexaFluor488, Donkey anti-rabbit Cy3 and Donkey anti-goat Cy3 (1/200, Jackson). The anti-Brn3 antibody marks the Brn3a, b and c, leading to the labeling of 80% of RGCs31 (link).
Sections were blocked with PGTx (0.2% gelatin and 0.25% Triton X-100 in PBS). Antibodies were diluted in PGTx: Rabbit anti-βGal (1/5000, Rockland), Goat anti-ChAT (1/200, Chemicon), Donkey anti-rabbit 488 and Donkey anti-goat Cy3 (1/500; Jackson Immunoresearch).
For retinal whole-mount immunostaining, retinas were first permeabilized in 1% Triton X-100 for 30 minutes, blocked in PHTx (0.1% Triton X-100 10% horse serum in PBS). Antibodies were diluted in PHTx: Rabbit anti-βGal (1/5000, Rockland), Goat anti-Brn3 (1/200, C-13, Santa Cruz), Rabbit anti-RBPMS (1/500, Phosphosolutions, # 1830), Donkey anti-rabbit AlexaFluor488, Donkey anti-chicken AlexaFluor488, Donkey anti-rabbit Cy3 and Donkey anti-goat Cy3 (1/200, Jackson). The anti-Brn3 antibody marks the Brn3a, b and c, leading to the labeling of 80% of RGCs31 (link).
3
Retinal Immunolabeling Protocol for Vertical Sections
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The immunolabeling protocol for vertical sections followed the procedures described in Molnar et al. (2016) (link) and Jo et al. (2017) (link). The retinas were fixed for 1 h in 4% paraformaldehyde, rinsed with PBS, dehydrated, and embedded in OCT compound mounting medium (Electron Microscopy Sciences); 12 μm thick cryosections were incubated in a blocking buffer (5% FBS and 0.3% Triton X-100 in 1X PBS) for 20 min. Primary antibodies (rabbit anti-TRPV4, 1:1000, LifeSpan Biosciences; rabbit anti-RBPMS, 1:500, PhosphoSolutions; mouse anti-Thy1.1, Sigma, 1:500; mouse SMI-32, 1:100, Covance; mouse anti-GFP, 1:500, Santa Cruz) were diluted in the diluent (2% BSA and 0.2% Triton X-100 in 1X PBS) and applied overnight at 4°C, followed by incubation in fluorophore-conjugated secondary antibodies (1:500; goat anti-mouse AlexaFluor 405, 488, or 647, goat anti-rabbit AlexaFuor 488 or 594, Life Technologies) for 1 h at RT. Images were acquired on an Olympus CV1200 confocal microscope using 20x (NA water) and 40x (0.9 NA water) objectives.
4
Fluorescent Immunohistochemistry of Brain Tissue
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Fluorescent immunohistochemistry (IHC) was performed on 20-μm cryosectioned PFA-fixed brain tissue as described in refs. 67 (link) and 84 (link)–87 (link, link, link). Primary antibodies were diluted in blocking buffer and incubated on tissue sections overnight at 4 °C. The following antibodies and dilutions were used: mouse anti-Brn3a (diluted 1:125, Millipore), rabbit anti-RFP (diluted 1:500, Rockland), rabbit anti-Opn4 [diluted 1:2,000, Dr. C.K. Chen’s laboratory (67 (link))], mouse anti- SMI32 (diluted 1:1,000, Covance), rabbit anti-RBPMS (diluted 1:500, PhosphoSolutions), rabbit anti-GFP (diluted 1:250, Invitrogen), mouse anti-NeuN (diluted 1:200, Millipore), rabbit anti-GFAP (1:1,000, DAkoCytomation), rabbit anti-Iba1 (1:500, Wako), mouse anti-GAD67 (diluted 1:500, Millipore), rabbit anti-calbindin (diluted 1:2,500, Swant), rabbit anti-calretinin (diluted 1:2,000, Swant), mouse anti-synaptophysin (diluted 1:500, SySy), and goat anti-NPNT (diluted 1:40, R&D systems). A minimum of three animals (per genotype and per age) were compared in all IHC experiments. Please reference SI Appendix for more details.
5
Immunohistochemistry of Thy1-CFP Retina
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Thy1-CFP mice were perfused transcardially with PBS followed by 4% paraformaldehyde (PFA) in PBS, then eyes were dissected and postfixed with 4% PFA in PBS overnight at 4 °C. Samples were cryoprotected by incubating in 30% sucrose in PBS for 48 h. Eyes were cryosectioned to 16-μm thickness. Tissue sections were blocked in 5% normal donkey serum and 0.3% Triton X-100 in PBS for 1 h and incubated with primary antibodies diluted in blocking buffer overnight at 4 °C, followed by 1-h incubation with secondary antibodies at room temperature. Primary antibodies used were rabbit anti-RBPMS 1:200 (PhosphoSolutions; cat# 1830) and chicken anti-GFP 1:1000 (Abcam; cat#13970).
6
Quantifying Retinal and Optic Nerve Cells
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Retinal flat-mounts were immunostained with rabbit anti-RBPMS (PhosphoSolutions) and DAPI nuclear stain. Total number of RBPMS-positive cells was estimated using stereology as previously described30 (link).
Optic nerves were postfixed in 4% PFA (in 0.1 M phosphate buffer, pH 7.4), placed in 1% osmium, dehydrated in ascending alcohol concentrations, and placed in 1% uranyl acetate in 100% ethanol for 1 h76 (link),77 (link). Optic nerves were embedded in epoxy resin mixture at 60 °C for 48 h and 1 µm sections prepared. Axon number was estimated using stereology, as previously described77 (link).
Optic nerves were postfixed in 4% PFA (in 0.1 M phosphate buffer, pH 7.4), placed in 1% osmium, dehydrated in ascending alcohol concentrations, and placed in 1% uranyl acetate in 100% ethanol for 1 h76 (link),77 (link). Optic nerves were embedded in epoxy resin mixture at 60 °C for 48 h and 1 µm sections prepared. Axon number was estimated using stereology, as previously described77 (link).
7
Multimodal Neurodevelopmental Marker Analysis
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Rabbit anti-Zfyve27 (Proteintech, 12680-1-AP, 1:500), Mouse Pan-Neurofascin (extracellular) (NeuroMab, 75–172, 1:50), Rabbit anti-Reticulon 4 (Novus Biologicals, NB100-56681, 1:250), Rabbit anti-RBPMS (Phosphosolutions, 1830-RBPMS, 1:500), Mouse anti-mCherry (ClonTech, 632543, 1:500), DAPI (ThermoFisher Scientific, D3751, 1:10,000), Sheep anti-GAP43 (kind donation from the Benowitz lab, 1:5000), mouse monoclonal anti-beta actin (C4) HRP conjugated (Santa Cruz, sc-47778, 1:5000), goat anti-rabbit IgG HRP conjugated (Sigma, A4914, 1:80000),Goat anti-rabbit 488 (ThermoFisher Scientific, A27034, 1:500), Goat anti-rabbit 568 (ThermoFisher Scientific, A-11011, 1:500), Goat anti-rabbit 647 (ThermoFisher Scientific, A27040, 1:500), Goat anti-mouse 647 (ThermoFisher Scientific, A-21235, 1:500), Goat anti-mouse 568 (ThermoFisher Scientific, A-11031, 1:500).
8
Immunostaining and Imaging Protocol for Retinal, Optic Nerve, and Brain Tissues
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After transcardial perfusion with saline (0.9% NaCl diluted in ddH2O) followed by 4% paraformaldehyde (PFA), the eyes, optic nerves and brain were harvested and post-fixed in 4% PFA for 24 hours. The eyes were then transferred to phosphate buffered saline (PBS) and the optic nerve and brain placed in 30% sucrose for cryoprotection. The optic nerve was sectioned using a sledge microtome, cutting longitudinally at 15 μm. The brain was sectioned coronally at 30 μm. All tissue sections were included for analysis. Retinas, optic nerves, and brain tissue were kept at 4° Celsius overnight using the following antibodies: rabbit-anti-RBPMS (PhosphoSolutions, 1:1000), to label RGCs; rabbit- or guinea pig-anti-GFP (SySy; 1:1000), to enhance GFP signal; rabbit-anti-p-S6 (Cell Signaling Technology; 1:250), to label phosphorylated S6 protein; mouse anti-SMI-32 (Sternberger monoclonals, 1:2000) to label alpha and other large soma RGC types; rabbit anti-melanopsin (Advanced Targeting Systems, 1:1000). For secondary detection, Alexa Fluor 488 goat anti-rabbit or anti-guinea pig (1:1000; Life Technologies), or Alexa Fluor 594 goat anti-rabbit (1:1000; Life Technologies) were used. Immunostained tissues were imaged with with an epifluorescence microscope (Zeiss Axio imager 2 with HR Zeiss camera, 10X and 20X objectives).
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