HUES6 hESC transducted with PODXL overexpressing virus and selected for blasticidin for 4 days. Anti‐RAC1 (1:200; Millipore, 05–389), Anti‐PODXL (1:50; R&D, MAB1658) antibodies were added in E8 medium, and cells were live stained at 4 °C for 2 hours with gentle shaking. Cells were rinsed with 1 × PBS two times and fixed with 4% paraformaldehyde for 10 min. Cells were blocked for 30 min (10% donkey serum, 0.5% Triton‐X100 in 1 × PBS) and then exposed to Donkey anti‐Mouse IgG (H+L) Highly Cross‐Adsorbed Secondary Antibody, Alexa Fluor 488 (1:400; Invitrogen, A‐21202), Donkey anti‐Goat IgG (H+L) or Cross‐Adsorbed Secondary Antibody, Alexa Fluor 555 (1:400; Invitrogen, A‐21432) antibodies in staining buffer [5% donkey serum (Millipore, S30), 0.1% Triton‐X100 in 1 × PBS] at RT for 2 hours with gentle shaking. Cells were washed three times with washing buffer [2% donkey serum (Millipore, S30), 0.05% Triton‐X100 in 1 × PBS]. 5 µL of Alexa Fluor 647 Phalloidin (Thermo Fisher Scientific, A22287) methanolic stock solution was diluted into 300 µL of staining solution for each reaction to be stained at 4 °C for 2 hours with gentle horizontal shaking. Nuclei were stained with DAPI (0.5 µg mL−1, D9542) for 10 min. Cells were mounted with ProLong Diamond Antifade mounting reagent (Thermo Fisher Scientific, P36965).
Mab1658
Manufactured by R&D Systems
Sourced in United States
MAB1658 is a monoclonal antibody product developed by R&D Systems. It is designed for use in various laboratory applications, but a detailed description of its core function cannot be provided in an unbiased and factual manner without extrapolation or interpretation.
Automatically generated - may contain errors
Lab products found in correlation
Mouse monoclonal anti gm130 clone 35 gm130 610822, by BD (2 mentions)
Mouse monoclonal anti γ tubulin clone gtu 88 t5326, by Merck Group (2 mentions)
Mouse monoclonal anti podxl clone 222328, by R&D Systems (2 mentions)
Bovine serum albumin a3311, by Merck Group (2 mentions)
Anti rac1, by Merck Group (1 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (1 mentions)
Prolong diamond anti fade mounting reagent, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Ibright 1500, by Thermo Fisher Scientific (1 mentions)
Complete mini protease inhibitor cocktail, by Roche (1 mentions)
4 protocols using mab1658
1
PODXL Overexpression in hESCs
2
Immunostaining of hPSCs in Matrigel
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hPSCs embedded in Matrigel were fixed with either 4% PFA at room temperature or ice-cold methanol at -20°C for 15 min. The samples were permeabilized with 0.3% Tx100 – 0.1 M glycine in PBS for 20 min. The primary antibodies mouse monoclonal anti-Oct3/4 (as above, 1:200), rabbit polyclonal anti-aPKC (as above, 1:200), rabbit polyclonal anti-PARD6B (as above, 1:200), mouse monoclonal anti-GM130 (clone 35/GM130; 610822; BD Biosciences; San Jose, US; 1:200), mouse monoclonal anti–γ–tubulin (clone GTU-88; T5326; Sigma, St.Louis, US; 1:500) and mouse monoclonal anti-PODXL (clone 222328; MAB1658; R&D Systems; Minneapolis, US; 1:200) were diluted in blocking buffer (1% Bovine Serum Albumin – A3311; Sigma-Aldrich; St. Louis, US - and 0.1% Tween) and incubated at 4°C over night. Samples were washed three times with PBS-0.1% Tween and incubated with AlexaFluor®-conjugated secondary antibodies (as above).
3
Immunostaining of hPSCs in Matrigel
4
Western Blot Analysis of Podocalyxin Expression
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Cells were lysed in lysis buffer containing 1% Triton X-100, 150 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, and complete mini protease inhibitor cocktail (Roche), and 20 mM DTT was added (samples for immunoblotting with mouse anti-podocalyxin (R&D Systems MAB1658) were not treated with DTT). NuPAGE LDS sample buffer (Invitrogen, NP0007) was added to the samples, which were heated to 95 °C for 5 min. Samples were loaded into 4–12% bis–tris gels next to SeeBlue™ Plus2 prestained protein standard as a protein marker in a running buffer prepared from 20 × NuPAGE MES buffer, ddH2O, and NuPAGE antioxidant. Samples were run for 45 min at 140 V. Gels were then transferred for blotting using a Novex iBlot dry blotting system (Invitrogen, cat# IB24001) and blocked in Tris-buffered saline (TBS) with 0.1% Tween-20 and 5% milk. The blots were incubated with primary antibodies at 4 °C overnight and washed in 0.1% Tween-20 in PBS, followed by incubation with secondary antibodies for 1 h at RT. After washing in 0.1% Tween-20 in PBS, the blots were developed using ECL substrate (Pierce, 32106) and detected using an iBright 1500 (Invitrogen) chemiluminescence imager.
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