hiPSC-derived sensory neurons were fixed with 4% paraformaldehyde for 10 min, washed twice with 0.1% Bovine Serum Albumin (BSA) in PBS, permeabilized with 0.2% Triton X-100 for 10 min and then blocked in 1% BSA for 1 h at room temperature. Fixed samples were incubated with primary antibodies against TUBB3 (1:1000, Covance, PRB-435P), Brn3a (1:25, Millipore, MAB1585), Peripherin (1:200, Millipore, AB1530), TRPV1 (1:100, Invitrogen, PA1-748), TRPM8 (1:100, NOVUS, NBP1-97311), Nav1.7 (1:100, NOVUS, NBP2-12904), TRKA (1:80, R&D SYSTEMS, MAB1751R), TRKB (1:80, R&D SYSTEMS, MAB3971), TRKC (1:100, R&D SYSTEMS, AF373), Neurofilament 200 (1:1000, Sigma, N4142), Isolectin B4 (1:1000, Thermo Fisher Scientific, I21411) over night at 4 °C. The following day, three washes were performed in 0.1% BSA in PBS, and the cells were incubated with appropriate secondary antibodies (1:500, Invitrogen) for 1 h at room temperature. All samples were incubated with DAPI (Sigma). A BZ-X810 microscope (Keyence) was used for recording the fluorescence images.
Prb 435p
Manufactured by Merck Group
Sourced in Germany
The PRB-435P is a laboratory equipment product manufactured by Merck Group. It is a precision Balance designed for accurate weighing of samples in a laboratory setting. The core function of the PRB-435P is to provide reliable and consistent measurements of weights up to 435 grams.
Automatically generated - may contain errors
Lab products found in correlation
Mab377, by Fortrea (3 mentions)
Prb 278p, by Merck Group (2 mentions)
Z0334, by Novus Biologicals (2 mentions)
Adi spa 223, by GeneTex (2 mentions)
Ab15766, by GeneTex (2 mentions)
Gtx62440, by Merck Group (2 mentions)
D9564, by Merck Group (2 mentions)
Gtx11268, by Fortrea (2 mentions)
Bz x810 microscope, by Keyence (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Isolectin b4, by Thermo Fisher Scientific (1 mentions)
Mab1585, by Merck Group (1 mentions)
Mab3971, by R&D Systems (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Mab5406, by Merck Group (1 mentions)
Ab152, by Merck Group (1 mentions)
Mab377, by Agilent Technologies (1 mentions)
In cell analyzer 2000, by GE Healthcare (1 mentions)
A2052, by Abcam (1 mentions)
Ab18465, by Merck Group (1 mentions)
9 protocols using prb 435p
1
Immunocytochemical Characterization of hiPSC-Derived Sensory Neurons
2
Immunofluorescence Staining of Neural Cells
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Cells were fixed in 4% paraformaldehyde (PFA) for 10 min, followed by washes in PBS at room temperature (RT). Cells were then blocked with 3% donkey serum in PBS containing 0.01% Triton X-100 for 1 hr at RT, incubated with primary antibodies overnight at 4°C, and then washed with PBS and incubated with secondary antibodies for 1 hr at RT. We used primary antibodies for GFAP (1:2,000; DAKO; Z0334), S100β (1:200; NOVUS; NB110–57478), ALDH1L1 (1:200; NeuroMab; 75–140), synapsin (1:1,000; SYSY; 106103), αB-crystallin (1:200; Enzo; ADI-SPA-223), MAP2 (1:500; GeneTex; GTX11268), Tuj1 (1:6,000; Covance; PRB-435P), NeuN (1:400; Millipore; MAB377), Pax6 (1:500; Covance; PRB-278P), Sox1 (1:500; Millipore; AB15766), Oligo2 (1:200; GeneTex; GTX62440), NG2 (1:500; Millipore; MAB5384), and NKX2.2 (1:50; DSHB; 745A5). Nuclei were stained with DAPI (1:6,000; Sigma; D9564).
3
Characterizing Neuronal and Astroglial Phenotypes
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Stylets (n = 3) were collected in the surgical room and their tip was immediately immersed in fixative solution and cell block preparation was performed using the Shandon™ cytoblock™ preparation system (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s protocol. Immuno-histofluorescence staining was performed on de-paraffinized BTIs 10 μm thick sections using antigen retrieval treatment and with secondary antibodies coupled with Alexa fluorochrome (A488 or A555). Neuronal and astroglial expression were identified using β3-tubulin (Covance, PRB435P) or NeuN (Millipore, MAB377) antibodies, and GFAP (DAKO, Z334) antibody, respectively. Further neuronal phenotypic characterization was investigated using antibody against: tyrosine hydroxylase (TH, Chemicon, AB152), vesicular glutamate transporter 1 for glutamatergic neurons (V-Glut1, Chemicon, MAB5502), glutamic acid decarboxylase-67 (GAD-67, Chemicon MAB5406) for GABA-ergic neurons. Sections were then counterstained with DAPI to label cell nuclei. Images were obtained using a fluorescence microscope Zeiss 5.1 (Zeiss, Iena, Germany) coupled to a digital camera and Axiovision 4.8 software (Zeiss).
4
Immunofluorescence Staining of Neural Cells
5
Immunocytochemistry of Neuronal Markers
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For QC, neurons were thawed, counted and seeded on 96-well plates (ibidi, m-plates) in NB/B27 medium supplemented with 10 mM RI. After 7 days, neurons were fixed with freshly thawed pre-warmed 4% PFA for 10-15 minutes. For the fixation of GABA, the PFA solution was supplemented with 0.04% glutaraldehyde. Cultures were blocked with 10% FBS in PBS + 0.1% Triton X-100 for one hour. The primary antibodies were diluted in 10% FBS in PBS + 0.1% Triton X-100 and incubated over night at 4 C. Afterward, adequate secondary antibodies were diluted in 10% FBS in PBS + 0.1% Triton X-100 and incubated for one hour. The following antibodies were used: BRN2 (ms IgG, Santa Cruz, sc-393324, 1:500; rb IgG, abcam 94977, 1:500), CTIP2 (rat IgG, abcam, ab18465, 1:500), GABA (ms IgG, Sigma, A0310, 1:1000; rb IgG, Sigma, A2052, 1:1000), SATB2 (ms IgG, abcam, ab92446, 1:100), TUJ1 (b3-tubulin, ms IgG, Covance, MMS-435P, 1:1000; rb IgG, Covance, PRB-435P, 1:2000; ck IgY, Millipore, AB9354, 1:500), vGlut2 (rb IgG, abcam ab84103, 1:500), FOXG1 (rb IgG, abcam, ab18259, 1:500). Images were acquired using the IN Cell Analyzer 2000 (GE Healthcare). Cellular markers were quantified automatically using the InCell Developer toolbox.
6
Immunofluorescence Staining of Stem Cell Markers
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Cells were fixed in 4% paraformaldehyde in PBS at 4 °C for 10 min, permeabilized at room temperature for 15 min in 1.0% Triton in PBS and blocked in 5% donkey serum with 0.1% Triton at room temperature for 30 min. The following primary antibodies and dilutions were used: goat anti-Nanog (R&D, AF1997), 1:200; mouse anti-Tra1-60 (Millipore, MAB4360), 1:100; mouse anti-human Nestin (Millipore, ABD69), goat anti-Sox2 (Santa Cruz, sc-17320), 1:200; rabbit anti-βIII-tubulin (Covance, PRB-435P), 1:200; mouse anti-MAP2AB (Sigma, M1406), 1:200; mouse anti-S100b (Sigma-Aldrich, S2532), 1:1000. Secondary antibodies were Alexa donkey anti-rabbit 488 (Jackson Immuno 711-545-152) and 568 (Life Technologies A10042), Alexa donkey anti-mouse 488 (Jackson Immuno 715-545-151) and 568 anti-mouse (Life Technologies A10037), and Alexa donkey anti-goat 488 (Jackson Immuno 705-545-147) and 568 (Jackson Immuno 705-605-147); all were used at 1:300. To visualize nuclei, slides were stained with 0.5 μg ml−1 DAPI (4′,6-diamidino-2-phenylindole) and then mounted with Vectashield.
7
Immunofluorescence Quantification of Neuronal Markers
8
Immunofluorescence and Immunochemistry Protocols
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Immunofluorescence and immunochemistry were performed as previously described (Cosset et al., 2015 (link), 2016 (link)). The following primary antibodies were used: mouse anti-neuronal nuclei-specific protein (NeuN) (Chemicon;MAB377), rabbit anti-βIII-tubulin (Covance;PRB435P), goat anti-ChaT (Chemicon; AB144P), rabbit anti-HB-9 (Abcam; ab922606), rabbit anti-ISLET1 (Abcam; ab22450), goat anti-GALR3 (Abcam), mouse anti-EV-71 (Abcam; ab36367), and mouse anti-PV-3 (Abcam; ab22450). Alexa Fluor (555 and 488)-labeled antibodies from goat or donkey against mouse, goat, or rabbit (Molecular Probes) were used as secondary antibodies. Cell nuclei were stained with DAPI (4, 6-diamidino-2-phenylindole). For IHC, biotin-conjugated anti-rabbit IgG or anti-goat IgG were used and developed using avidin-biotin peroxidase detection system (Vector Labs) with 3,3′-diaminobenzidine substrate (DAB, Sigma-Aldrich) after 2 min of incubation.
9
Characterization of Pluripotent Stem Cells
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For alkaline phosphatase staining, iPSCs were fixed in a citrate–acetone–formaldehyde solution and then stained with alkaline phosphatase staining solution (Naphthol/fast red violet, Sigma). Cell images were captured using an Olympus microscope (IX51, Olympus, Tokyo, Japan).
For immunofluorescence staining, cells were fixed in 4% formaldehyde and then permeabilized with phosphate-buffered saline containing 0.1% Triton X-100. After blocking with 3% bovine serum albumin, cells were incubated with the following primary antibodies: OCT4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-9081, 1:200), NANOG (Santa Cruz Biotechnology, sc-33759, 1:100), TRA-1-81 (Chemicon, Santa Cruz, CA, USA, MAB4381, 1:100), TRA-1-60 (Chemicon, MAB4360, 1:100), SSEA3 (R&D Systems, Minneapolis, MN, USA, MAB1434, 1:100), SSEA4 (R&D Systems, MAB1435, 1:100), DESMIN (Chemicon, AB907, 1:50), α-smooth muscle actin (Sigma-Aldrich, Carlsbad, CA, USA, A5228, 1:400), FOXA2 (Abcam, Cambridge, MA, USA, ab40874, 1:500), SOX17 (R&D Systems, MAB1924, 1:100), TUJ1 (Covance, Munich, Germany, PRB-435P, 1:500) and NESTIN (Chemicon MAB5326, 1:100). DAPI (4',6-diamidino-2-phenylindole) was used for nuclear counterstaining. Chamber slides were observed with an Axiovert 200M microscope (Carl Zeiss, Gottingen, Germany) or an Olympus microscope (Olympus).
For immunofluorescence staining, cells were fixed in 4% formaldehyde and then permeabilized with phosphate-buffered saline containing 0.1% Triton X-100. After blocking with 3% bovine serum albumin, cells were incubated with the following primary antibodies: OCT4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-9081, 1:200), NANOG (Santa Cruz Biotechnology, sc-33759, 1:100), TRA-1-81 (Chemicon, Santa Cruz, CA, USA, MAB4381, 1:100), TRA-1-60 (Chemicon, MAB4360, 1:100), SSEA3 (R&D Systems, Minneapolis, MN, USA, MAB1434, 1:100), SSEA4 (R&D Systems, MAB1435, 1:100), DESMIN (Chemicon, AB907, 1:50), α-smooth muscle actin (Sigma-Aldrich, Carlsbad, CA, USA, A5228, 1:400), FOXA2 (Abcam, Cambridge, MA, USA, ab40874, 1:500), SOX17 (R&D Systems, MAB1924, 1:100), TUJ1 (Covance, Munich, Germany, PRB-435P, 1:500) and NESTIN (Chemicon MAB5326, 1:100). DAPI (4',6-diamidino-2-phenylindole) was used for nuclear counterstaining. Chamber slides were observed with an Axiovert 200M microscope (Carl Zeiss, Gottingen, Germany) or an Olympus microscope (Olympus).
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