Human TM cells and sectioned tissues were washed with PBS, fixed with 4% paraformaldehyde (CWBIO) for 12 min, permeabilized with 0.3% TritonX-100 (CWBIO) and blocked with 5% albumin serum (BSA) for 1 h. The samples were then incubated with primary antibodies at 4°C overnight, followed by 1-h incubation with Alexa Fluor 488 (A21206; Thermo) or 546 (A10036; Thermo) secondary antibodies. Nuclei were stained with 4’, 6-diamidino-2-phenylindole (Vector Laboratories, Peterborough, United Kingdom). Images were captured with a Zeiss or Leica confocal imaging system (Carl Zeiss; Hertfordshire, United Kingdom). Primary antibody myocilin (sc-515500, Santa Cruz), RBPMS(GTX118619, GeneTex) and KDEL (ab176333, abcam) were diluted at a concentration of 1:200.
Rbpms
Manufactured by GeneTex
Sourced in United States
RBPMS is a laboratory equipment product. It functions as an RNA-binding protein that is involved in the regulation of gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Myocilin sc 515500, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Alexa fluor 594 conjugated anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Application suite x 3, by Leica (1 mentions)
Dmi8 confocal microscope, by Leica (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Cryotome, by Leica (1 mentions)
Neuronal nuclei (neun), by Thermo Fisher Scientific (1 mentions)
Gsdmd, by Santa Cruz Biotechnology (1 mentions)
Il 1b, by Cell Signaling Technology (1 mentions)
Nlrp1, by Novus Biologicals (1 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (1 mentions)
Cd11b, by BioLegend (1 mentions)
Nlrp3, by Adipogen (1 mentions)
Fibronectin, by Abcam (1 mentions)
Grp94, by Santa Cruz Biotechnology (1 mentions)
Collagen 1, by Novus Biologicals (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
7 protocols using rbpms
1
Immunofluorescent Labeling of Human TM Cells
2
Retinal Ganglion Cell Immunolabeling
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Freshly isolated eyes were immersion fixed in 4% paraformaldehyde for 30 min then cryoprotected in 30% sucrose with 0.02% sodium azide. Retinas were dissected out and vitreous removed. Immunolabeling included washes in 0.1 M PBS, then incubation in blocking solution (5% donkey serum, 1% Triton X-100 in 0.1 M PBS) for 1 h, primary antibody (diluted in 0.5% bovine serum albumin, 0.9% NaCl, 1% Triton X-100 in 0.1 M PBS) for 48–72 h, washes in 0.1 M PBS, blocking solution for 30 min, secondary antibody incubation for 18 h, washes, DAPI labeling, then coverslipping with Fluoromount-G. RBPMS (RNA binding protein, multiple splicing) primary antibody (Genetex, Irvine, CA) to label RGCs (1:250) and secondary antibody from Jackson ImmunoResearch (West Grove, PA) Alexa Fluor 594-conjugated anti-rabbit IgG (1:250) were used. Images were collected on a Leica DMi8 confocal microscope integrated with Leica application Suite X 3.1.1.15751 (Leica Microsystems, Buffalo Grove, IL, USA). Figures were created using Adobe Illustrator, Adobe Creative Cloud version 2017.
3
Immunofluorescence Imaging of Autophagy Markers
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TM cells were plated on coverslips in 24-well plates and grown overnight to 50%–60% confluence. Cells were fixed with 4% paraformaldehyde (PFA) for 12 min at room temperature and permeabilized with 0.3% Triton X-100 (CWBIO) for 15 min. Subsequently, cells were blocked with 2% BSA for 1 h at room temperature. Then, cells were incubated with primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies for 1 h. Finally, cells were stained with 4,6-diamidino-2-phenylindole (DAPI). Fluorescence images were captured using Zeiss or Leica confocal imaging systems (Carl Zeiss; Leica). The following antibodies were used: p62 (5114, Cell Signaling Technology), LC3B (43566, Cell Signaling Technology), KDEL (ab176333, Abcam), RBPMS (GTX118619, GeneTex), Alexa Fluor 488 (A21206; Thermo), and Alexa Fluor 546 (A10036; Thermo).
4
Immunohistochemical Analysis of Retinal Cells
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Eyes were enucleated, fixed in 4% paraformaldehyde for 30 min, and cryoprotected with 30% sucrose. Whole eyes were frozen and then sectioned to a thickness of 10 μm on a Leica cryotome (Germany). After washing, permeabilization and blocking, sections were incubated with a primary antibody for 4–16 h. Retinal flat-mounts were incubated with primary antibodies for 3–5 days at 4°C to ensure even staining across retinal layers. AlexaFluor dye-labeled and secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA) were applied for imaging. The following commercially available antibodies were used: neuronal Brn3a (Santa Cruz), class III β-tubulin (TUJ-1, Covance ID # MMS-435P), RBPMS (GeneTex ID# GTX118619), NeuN (Invitrogen ID#702022); glutamine synthetase (GeneTex ID# GTX109121); IL-1b (Cell Signaling ID#8689); Iba1 (Wako/FUJI ID# 019-19741), GFAP (Dako, cat#z0334), CD11b (Biolegend ID#101218), Casp1 (Novus Biologicals ID#NB100-56565R), Casp11 (Novus Biologicals ID# NB120-10454), ASC (Adipogen ID#AG25b-0006), NLRP1 (Novus Biologicals ID #NB100-56148), NLRP3 (Adipogen ID# AG20b-0014-C100), Aim2 (Boster Biological Technology ID#PB9683), GSDMD (Santa Cruz ID#sc-393656). Species-specific secondary fluorescence AlexaFluor dye-conjugated antibodies for confocal microscopy were purchased from Invitrogen/Molecular Probes, USA.
5
Antibody Sourcing for ER Stress Pathway
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Antibodies were purchased from the following sources; fibronectin (catalog # Ab2413, Abcam), KDEL (catalog # Ab12223, Abcam), collagen I (catalog # NB600-408, Novus Biologicals), ATF4 (catalog # SC-200, Santa Cruz Biotechnology), CHOP (catalog # 13172, Novus Biologicals), GRP78 (catalog # ab21685, Abcam), GRP94 (catalog # 11402, Santa Cruz Biotechnology), Caspase 3 (catalog # 9662, Cell signaling technology), Cleaved PARP (catalog # 9541, Cell signaling technology), puromycin (catalog #A11138, Gibco, Life Technology), RBPMS (catalog # 118619, Gene Tex), cleaved Caspase 3 (catalog # 9661, Cell signaling technology), GAPDH (catalog # 3683, Cell signaling technology), and β-Actin (catalog # 4970, Cell signaling technology, Danvers, MA, USA). For immunoprecipitation studies, a different CHOP (sc-7351, Santa Cruz Biotechnology) was used. All adenoviral vectors used in this study were obtained from ViraQuest Inc. (North Liberty, IA, USA). Lentiviral vectors expressing ATF4 or CHOP were obtained from VectorBuilder Inc.
6
Quantifying Retinal Ganglion Cell Survival
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RGC counting was performed as previously described [24 (link)]. The retinas were dissected, and four radial cuts were made to allow the dissected retinas to lie flat. Then, the retinas were incubated with tubulin‐βIII (1:500, mouse polyclonal; Biolegend, #801202, San Diego, America) and RBPMS (1:500, rabbit polyclonal; GeneTex, #GTX118619, Southern California, America) antibodies overnight at 4 °C. The following day, the cells were incubated with Alexa Fluor 555‐conjugated secondary antibody (1:500, Abcam, #ab150074, Cambridge, England) to rabbit IgG and Alexa Fluor 488‐conjugated secondary antibody (1:500, Abcam, #ab150105, Cambridge, England) to mouse IgG. The images were captured with light microscopy (Leica DM6B). Images of each of the four flaps of each retina were taken 1 mm and 2 mm from the optic nerve head. The number of RGCs in each photo was averaged to estimate the overall RGC survival per square millimetre.
7
Immunostaining of Retinal Sections
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Immunostaining of retinal sections was performed as described [35 (link)]. Mouse eyes were enucleated, embedded in optimal cutting temperature mounting medium (Tissue-Tek, Torrance, CA, USA), frozen on dry ice, and cryostat sectioned (10 µm). Slides were incubated overnight at 4 °C with anti-ADAM17 (1:500; LSBio, Seatle, WA, USA), anti-CD31 (10 µg/mL; R&D Systems, Minneapolis, MN, USA), and anti-RNA binding protein with multiple splicing (RBPMS) (1:500; GeneTex, Zeeland, MI, USA) antibodies, followed by incubation with appropriate fluorescence-conjugated secondary antibodies (Life Technologies, Eugene, OR, USA). Secondary antibody controls (no primary antibody) were included in each experiment. Sections were mounted using Fluoroshield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclei (Sigma-Aldrich, St. Louis, MO, USA) and examined for epifluorescence using a Zeiss Axioplan-2 microscope (Carl Zeiss, Göttingen, Germany) equipped with the Axiovision program (version 4.7; Carl Zeiss).
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