Immunofluorescence experiments were carried out following our previously reported protocols [37 (link)]. Briefly, cells growing on the glass slide were fixed with 4% paraformaldehyde for 15 min and permeabilized using 0.25% Triton X-100 diluted in PBS for 10 min at room temperature. To block unspecific epitopes, cells were incubated with PBS containing 1% BSA and 0.1% Tween-20 for 1 h. To evaluated the pluripotency of hMBSCs, we used the following antibodies: rabbit anti-OCT4 (5 μg/ml, Abcam, Nanchang, China), mouse anti-SSEA-4 (15 μg/ml, Abcam), and rabbit anti-Nanog (1 : 200, Abcam). The fixed cells were incubated overnight at 4°C with primary antibodies followed by incubation with secondary donkey anti-mouse or anti-rabbit antibodies conjugated to either Alexa Fluor 488 or Alexa Fluor 568 (Jackson, Nanchang, China). Nuclei were counterstained with DAPI (Thermo Fisher).
Rabbit anti oct4
Manufactured by Abcam
Sourced in China, United States
Rabbit anti-OCT4 is a primary antibody that specifically recognizes the OCT4 (also known as POU5F1) protein, a transcription factor that plays a crucial role in the maintenance of pluripotency and self-renewal in embryonic stem cells. This antibody can be used in various immunoassays to detect and quantify the expression of OCT4 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti nanog, by Abcam (10 mentions)
Rabbit anti sox2, by Abcam (7 mentions)
Mouse anti ssea4, by Abcam (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Rabbit anti sox2, by Thermo Fisher Scientific (4 mentions)
Alexa fluor 568, by Jackson ImmunoResearch (2 mentions)
Alexa fluor 488, by Jackson ImmunoResearch (2 mentions)
Rabbit anti e cadherin, by Abcam (2 mentions)
Mouse anti sox2, by R&D Systems (2 mentions)
Mouse anti α tubulin, by Santa Cruz Biotechnology (2 mentions)
Mouse anti lama c, by Santa Cruz Biotechnology (2 mentions)
Mouse anti sox2, by Abcam (2 mentions)
Mouse anti β actin, by Merck Group (2 mentions)
Protein a g magnetic bead, by Thermo Fisher Scientific (2 mentions)
Rabbit anti integrin α5, by Abcam (2 mentions)
Rabbit anti integrin β1, by Abcam (2 mentions)
Rabbit anti vinculin, by Abcam (2 mentions)
Rabbit anti vimentin, by Cell Signaling Technology (2 mentions)
Mouse anti tra 1 60, by Thermo Fisher Scientific (2 mentions)
Rat anti k8, by Developmental Studies Hybridoma Bank (2 mentions)
24 protocols using rabbit anti oct4
1
Evaluating Pluripotency Markers in hMBSCs
2
Human iPSC-Derived Microglia Generation
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Three separate human iPSC lines were used in these studies to ensure that the
successful generation of microglia could be reproduced using multiple iPSCs.
Human iPSC line NCRM-1 was obtained from the National Institutes of Health Stem
Cell Resource (Bethesda, MD, USA). Human iPSC lines CS52iALS-C9n6.ISOxx
(referred to as “CS52” hereafter for sake of brevity) and CS29iALS-C9n1.ISOxx
(“CS29” hereafter), corresponding to corrected, isogenic lines derived from two
distinct parental lines generated from ALS patients with
C9orf72 gene mutations, were obtained from Cedars-Sinai
(Los Angeles, CA, USA) (Thiry et al., 2022 (link)). Undifferentiated state of human iPSCs was
assessed by testing for expression of the stem cell markers NANOG and OCT4 using
rabbit anti-NANOG (1/1,000; Abcam; Cambridge, UK; Cat. No. ab21624) and rabbit
anti-OCT4 (1 µg/ml; Abcam; Cat. No. ab19857) or goat anti-OCT3/4 (1/500; Santa
Cruz Biotechnology; Dallas, TX, USA, Cat. No. sc-8628) antibodies, as described
(Chen et al.,
2021 ).
successful generation of microglia could be reproduced using multiple iPSCs.
Human iPSC line NCRM-1 was obtained from the National Institutes of Health Stem
Cell Resource (Bethesda, MD, USA). Human iPSC lines CS52iALS-C9n6.ISOxx
(referred to as “CS52” hereafter for sake of brevity) and CS29iALS-C9n1.ISOxx
(“CS29” hereafter), corresponding to corrected, isogenic lines derived from two
distinct parental lines generated from ALS patients with
C9orf72 gene mutations, were obtained from Cedars-Sinai
(Los Angeles, CA, USA) (Thiry et al., 2022 (link)). Undifferentiated state of human iPSCs was
assessed by testing for expression of the stem cell markers NANOG and OCT4 using
rabbit anti-NANOG (1/1,000; Abcam; Cambridge, UK; Cat. No. ab21624) and rabbit
anti-OCT4 (1 µg/ml; Abcam; Cat. No. ab19857) or goat anti-OCT3/4 (1/500; Santa
Cruz Biotechnology; Dallas, TX, USA, Cat. No. sc-8628) antibodies, as described
(
2021
3
Western Blot Analysis of Stem Cell Markers
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Cells were lysed in RIPA lysis buffer (Solarbio, China) supplemented with 1% PMSF (Solarbio, China). Protein concentration was determined using Enhanced BCA Protein Assay Kit (Beyotime, China). Equal amount of protein sample was separated on 10 % SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Solarbio, China). The membranes were blocked in 5% BSA buffer (Solarbio, China) and then incubated with primary antibodies (Rabbit anti-Oct4, 1:1000, Abcam; Rabbit anti-Nanog, 1:1000, Abcam; Rabbit anti-N-cadherin, 1:5000, Abcam; Rabbit anti-E-cadherin, 1:10000, Abcam; Rabbit anti-vimentin, 1:1000, Abcam; Rabbit Anti-Snail, 1:1000, Cell Signaling; Rabbit Anti-GAPDH, 1:10000, Abcam) overnight at 4°C or for 2 h at room temperature. After three times of washes, the membranes were incubated with horseradish peroxidase-conjugated secondary goat anti-rabbit antibodies (1:4000; Biosharp, China) for 1 h at room temperature according to the manufacturer's instructions. The detection of immunocomplexes was performed with an enhanced chemiluminescence system (NEN Life Science Products, USA). The experiments were carried out on three separate occasions.
4
Immunofluorescence Characterization of Stem Cells
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Cells were fixed with 4% paraformaldehyde for 15 min at room temperature (RT) and permeabilized for 15 min in 0.1% NP-40 in phosphate-buffered saline (PBS). Cells were blocked using 1% of normal goat serum for 1 h at RT. The following antibodies were used: rabbit anti-OCT4 (Abcam), mouse anti-TRA-1-60 (Millipore), rabbit anti-NANOG (Stemgent), mouse anti-SSEA-4 (Millipore), rabbit anti-TH (Calbiochem), mouse anti-SOX2 (R&D Systems), mouse anti-mouse anti-Nestin (Santa Cruz), mouse anti-Pax6 (Santa Cruz). Corresponding Alexa Fluor-conjugated secondary antibodies were from Thermo Fisher. To label nuclei, coverslips were mounted using DAPI Fluoromount-G (SouthernBiotech). Images were acquired using a confocal microscope (Zeiss LSM710) operated by the ZEN software package. ImageJ (Schneider et al., 2012 (link)) and Adobe Photoshop CS6 (Adobe, United States) software were used for final image processing.
5
Immunocytochemical Staining Protocol for Stem Cell Markers
6
Immunofluorescence Staining of H9-ESCs
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H9‐ESCs seeded on the 1% Matrigel‐coated round coverslip in 24‐well plate on day 4 were fixed with 4% paraformaldehyde (PFA, Servicebio, Wuhan, Hubei, China) for 30 min at room temperature. After treating with 0.2% Triton X‐100 and 10% donkey serum and incubating at room temperature for 30 min, cells were incubated with the following primary antibodies in 10% donkey serum overnight at 4 °C: rabbit anti‐NANOG (1/200, Abcam, UK), rabbit anti‐OCT4 (1 : 250, Abcam) and rabbit anti‐SOX2 (1/200, Abcam). Subsequently, cells were incubated for 1 h with AlexaFluor‐568‐conjugated secondary antibody (1 : 400, Thermo) in 0.01 m phosphate‐buffered saline (PBS, Gibco, Grand Island, NY, USA) at room temperature. After washing cells with PBS for three times (5 min), the nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI, 1 : 500, Sigma, Saint Louis, MO, USA) in PBS for 10 min at room temperature. Finally, images were captured at 200× magnification using a Zeiss Axio Imager Z2 microscope (Zeiss, Oberkochen, Germany) that was equipped with tissuefaxs software (TissueGnostics GmbH, Vienna, Austria) and prepared using imagej software (Version 1.52a, NIH, Bethesda, MD, USA) and prism 8 for macOS (Version 8.4.0, GraphPad Software, San Diego, CA, USA).
7
Pluripotency and Epithelial-Mesenchymal Transition Markers
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Primary antibodies used in this study included rabbit anti-SOX-2 (1:1000 dilution, Abcam, Cambridge, UK), rabbit anti-Nanog (1:1000 dilution, CST, Danvers, Massachusetts, USA), rabbit anti-OCT-4 (1:1000 dilution, Abcam), rabbit anti-pSmad2/3 (1:500 dilution, Abcam), rabbit anti-Snail (1:500 dilution, Abcam), rabbit anti-E-cadherin (1:500 dilution, Abcam), rabbit anti-TGF-β1(1:1000 dilution, Abcam), rabbit anti-N-cadherin (1:1000 dilution, Wanleibio), rabbit anti-Slug (1:1000 dilution, Wanleibio) and mouse anti-β-actin (1:1000 dilution, ZSGB-BIO, Beijing, China).
8
Immunostaining of Pluripotent Stem Cells
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E14 ES cells were fixed for 20 min with a chilled mix of 95% methanol/5% acetic acid that was maintained at −20°C, permeabilized with chilled PBS-Triton for 15 min, and blocked with PBS and 1% BSA for 30–60 min. Samples were incubated with primary antibodies rabbit anti-Sox2 (1:200; Life Technologies) and rabbit anti-Oct4 (1:500; Abcam, ab19857) in PBS and 1% BSA overnight at 4°C. Samples were washed twice in PBS and then incubated with Alexa 555 anti-rabbit (1:1000; Life Technologies, catalog no. 481400) in PBS and 1% BSA for 45–60 min followed by three washes with 0.1% PBS-Tween, incubation with 2 ng/mL DAPI, three washes with PBS and 0.1% Tween, and two washes with PBS.
9
Protein Expression Analysis in Stem Cells
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Total proteins were extracted from the REFs and RiPSCs-6F/CR using the total protein extraction kit (cat. no. P0028; Beyotime Institute of Biotechnology) and quantified using a BCA protein assay kit (cat. no. P0010; Beyotime Institute of Biotechnology). Equivalent quantities of protein (40 µg/sample) were separated by SDS-PAGE on 10% gels and electroblotted onto PVDF membranes (0.45 µM; MilliporeSigma). The membranes were blocked with 5% skimmed milk powder diluted in TBS-0.05% Tween at room temperature for 1 h and immunoblotted overnight at 4°C with the following primary antibodies: Rabbit anti-OCT4 (1:200; Abcam), rabbit anti-Nanog (1:300), rabbit anti-Sox2 (1:500; both from Cell Signaling Technology, Inc.) and mouse anti-β-actin (1:1,000; Affinity Biosciences). Subsequently, the membranes were incubated with a HRP-labeled secondary antibody (1:5,000; cat. no. 111-035-003; Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature. The protein bands were then visualized using Clarity Western ECL Substrate (Bio-Rad Laboratories, Inc.) and were semi-quantified using Quantity One software version 1-D (Bio-Rad Laboratories, Inc.).
10
Immunoprecipitation of OCT4 Protein
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