Cells were grown on precoated glass coverslips (Neuvitro Corporation, Vancouver, WA) and fixed in 4% paraformaldehyde solution in phosphate-buffered saline. Cells were blocked with bovine serum albumin and normal serum from the species specific to the primary antibodies. Primary antibodies used were mouse IgG2b anti-TuJ1 (Sigma), chicken anti–glial fibrillary acidic protein (GFAP; Sigma), rabbit anti-S100ß (Dako Corporation, Carpinteria, CA), guinea pig anti-vGluT1 (Synaptic Systems, Göttingen, Germany), mouse anti–Tra-1–60 (Thermo Fisher Scientific), goat anti-Oct3/4 (Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit anti-Sox2 (Millipore). Fluorophore-conjugated donkey secondary antibodies against the primary antibody host species IgG were from Thermo Fisher Scientific. Nuclei were counterstained with DAPI (Thermo Fisher Scientific), and cells were mounted using Prolong Gold media (Thermo Fisher Scientific). Cell counts were performed in ImageJ, and values for each genotype and patient background were determined by the average of 3 fields per coverslip and 3 coverslips.
Goat anti oct3 4
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Goat anti-Oct3/4 is a primary antibody that specifically binds to the Oct3/4 (Octamer-binding transcription factor 3/4) protein. Oct3/4 is a key regulator of embryonic stem cell pluripotency and is commonly used as a marker for undifferentiated stem cells.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Tcs sp5 laser scanning microscope, by Leica (2 mentions)
Goat anti sox2, by Santa Cruz Biotechnology (2 mentions)
Mouse anti tra 1 81, by Merck Group (2 mentions)
Mouse anti ssea4, by Cell Signaling Technology (2 mentions)
Rabbit anti foxa2, by Cell Signaling Technology (2 mentions)
Goat anti nanog, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti sox2, by Santa Cruz Biotechnology (2 mentions)
Guinea pig anti vglut1, by Synaptic Systems (1 mentions)
Rabbit anti sox2, by Merck Group (1 mentions)
Mouse anti tra 1 60, by Thermo Fisher Scientific (1 mentions)
Rabbit anti oct4, by Abcam (1 mentions)
Las v3, by Leica (1 mentions)
Alexa fluor 488 donkey anti goat, by Thermo Fisher Scientific (1 mentions)
Bsa pbs, by Merck Group (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions)
Mouse anti ssea4, by Abcam (1 mentions)
Mouse anti tra 1 60, by Abcam (1 mentions)
Mouse anti glucagon, by Merck Group (1 mentions)
Goat anti pdx1, by R&D Systems (1 mentions)
9 protocols using goat anti oct3 4
1
Immunocytochemical Characterization of Cells
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
Immunocytochemistry of hiPSCs
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hiPSCs growing for 5 days on 24 well plates were washed three times with DPBS (Life Technologies), fixed in PFA 4% for 20 min at RT, permeabilized with 0.1% Triton X-100 in PBS for 15 min at RT and blocked overnight at 4 °C in 4% BSA/PBS (Sigma Aldrich). The primary antibodies were incubated 1 h at RT, followed by incubation with the secondary antibodies. The following primary antibodies were used: goat anti-Oct3/4 (Santa Cruz Biotechnology Inc., Dallas, TX, USA), mouse anti-SSEA4 (Abcam, Cambridge, UK), mouse anti-TRA-1-60 (Abcam), mouse anti-KLF4 (Merck Millipore). Upon incubation with the appropriate secondary antibodies (AlexaFluor® 488 Donkey anti-goat (Thermo Fisher Scientific, Whaltham, MA, USA), H&L (FITC) and Goat anti-Mouse, both from Abcam) cells were stained with DAPI (Life Technologies), fluorescence was observed in a Leica microscope and images were taken using LAS V3.8 software (Leica).
4
Immunofluorescence Analysis of Stem Cell Markers
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Cells were fixed with 4% paraformaldehyde for 30 min and then permeabilized in PBS containing 0.2% Triton X-100. Cells were blocked with PBS containing 3% BSA, and incubated with primary antibodies overnight at 4 °C. Then the cells were incubated with the secondary antibodies for 1 h at room temperature after washing with PBS. Images were acquired on a TCS SP5 laser-scanning microscope (Leica). The following antibodies and dilutions were used: goat anti-OCT-3/4 (1:500, Cat #sc-8628, Santa Cruz), goat anti-SOX2 (1:500, Cat #sc-17320, Santa Cruz), mouse anti-TRA-1-81 (1:50, Cat #MAB4381, Millipore), mouse anti-SSEA4 (1:500, Cat #4755, Cell Signaling), rabbit anti-FOXA2 (1:250, Cat #8186, Cell Signaling), goat anti-SOX17 (1:500, Cat #GT15094, Acris/Novus), goat anti-PDX1 (1:500, Cat #AF2419, R&D Systems), rabbit anti-NKX6.1 (1:300, Cat #NBP1-82553, Acris/Novus), guinea pig anti-C-Peptide (1:100, Cat #ab30477, Abcam), mouse anti-Glucagon (1:500, Cat #G2654, Sigma), rat anti-Somatostatin (1:300, Cat #MA5-16987, Invitrogen).
5
Immunofluorescence Staining of Stem Cells
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Undifferentiated PSCs and AECs were rinsed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (Sigma) for 20 min at room temperature. After washing with PBS, cells were permeabilized with 0.5% saponin (Sigma) in PBS containing 1% bovine serum albumin for 10 min and then blocked with 10% normal rabbit or donkey serum for 30 min. The following primary and secondary antibodies were used: goat anti-OCT3/4 (Santa Cruz), mouse anti-CPM (Abcam), rabbit anti-NKX2.1 (Abcam), mouse anti-EPCAM (Santa Cruz), rabbit anti-goat Alexa Flour 594 (Invitrogen), goat anti-mouse Alexa Fluor 594 (Invitrogen), donkey anti-mouse Alexa Fluor 594 (Invitrogen), and donkey anti-rabbit Alexa Fluor 488 (Invitrogen). Nuclei were stained with prolong gold DAPI with antifade (Invitrogen). Immunofluorescence signals were observed and photographed with an Olympus IX51 microscope equipped with a photometrix Cool Snap HQ2 camera using Image-Pro 3DA vs. 6.0. software.
6
Immunofluorescence Characterization of Stem Cells
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Cells were fixed with 4% paraformaldehyde for 30 min and then permeabilized in PBS containing 0.2% Triton X-100. Cells were blocked with PBS containing 3% BSA, and incubated with primary antibodies overnight at 4 °C. Then secondary antibodies were incubated for 1 h at room temperature after washing with PBS. Images were acquired on a TCS SP5 laser-scanning microscope (Leica). The following antibodies and dilutions were used: goat anti-OCT-3/4 (1:500, #sc-8628, Santa Cruz), goat anti-SOX2 (1:500, #sc-17320, Santa Cruz), mouse anti-TRA-1-60 (1:1000, #4746, Cell Signaling), mouse anti-TRA-1-81 (1:50, MAB4381, Millipore), mouse anti-SSEA4 (1:500, #4755, Cell Signaling), rabbit anti-FOXA2 (1:250, #8186, Cell Signaling), goat anti-SOX17 antibody (1:500, #GT15094, Acris/Novus), goat anti-PDX1 antibody (1:500, #AF2419, R&D Systems).
7
Western Blot Analysis of Stem Cell Markers
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Protein lysate of all the samples was prepared using RIPA buffer (Thermoscientific) containing protease inhibitors. Protein concentration was determined by Microplate BCA Protein Assay kit (Thermo Scientific), a total of 25 μg each protein sample was separated using 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE, Mini Protean, BioRad) and transferred onto polyvinylidene difluoride membranes (PVDF, Millipore, USA). Membranes were then incubated with primary antibodies of goat anti-Oct-3/4 (43–50 kDa, 1:200, Santa Cruz), rabbit anti-Sox-2 (34 kDa, 1:200, Santa Cruz), goat anti-Nanog (35 kDa, 1:200, Santa Cruz), rabbit anti-Bax (22 kDa, 1:1000, Enzo, NY, USA), rabbit anti-Bcl-2 (28 kDa, 1:1000, Cell Signalling, MA, USA), mouse anti-p53 (53 kDa, 1:200, Santa Cruz), rabbit anti-p21 (21 kDa, 1:200, Santa Cruz) and rabbit anti-β actin (45 kDa, 1:1000, Cell Signalling) for overnight at 4°C followed by incubation with horseradish peroxidase (HRP)-conjugated donkey anti-goat IgG (1:10000, Santa Cruz), goat anti-rabbit IgG (1:10000, Santa Cruz) and goat anti-mouse IgG (1:10000, Santa Cruz) secondary antibodies for 1 h at room temperature. Immunoreactivity was detected by enhanced chemiluminescence (ECL; Supersignal, West Pico Chemiluminescent substrate, PIERCE, IL, USA) and exposed to x-ray films.
8
Immunocytochemical Characterization of Stem Cells
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Immunocytochemical staining was performed by fixing cells with 3.7% formaldehyde for 30 min and permeabilized with 0.25% Triton X‐100 for 10 min at room temperature. After blocking with 1% bovine serum albumin (BSA) in DPBS for 1 h, cells were incubated with primary antibodies, such as goat anti‐Oct‐3/4 (1:200, Santa Cruz Biotechnology, CA), rabbit anti‐Sox‐2 (1:200, Santa Cruz Biotechnology), goat anti‐Nanog (1:200, Santa Cruz Biotechnology), goat anti‐human serum albumin (ALB, 1:200, Santa Cruz Biotechnology) and goat anti‐hepatocyte nuclear factor 1‐alpha (HNF‐1α, 1:200, Santa Cruz Biotechnology) for overnight at 4°C followed by incubation with CruzFluor™ 594 conjugated donkey anti‐goat IgG (1:200, Santa Cruz Biotechnology) or donkey anti‐rabbit IgG (1:200, Santa Cruz Biotechnology) secondary antibodies for 45 min at 37°C. The nuclei of cells were counterstained with 1 µg/ml 4′,6‐diamidino‐2‐phenylindole (DAPI) for 5 min and images were taken using fluorescence microscope (Leica, Wetzlar, Germany).
9
Antibody Characterization for Cell Research
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The primary antibodies used included mouse anti-Nup153, ascites fluid (gift from B. Burke), rabbit anti-Nup153 (raised against hNup153-GST, amino acids 1300–1399), rabbit anti-Parp (9542, Cell Signaling), mouse anti-Oct-3/4 (sc-5279, Santa Cruz Biotechnology), goat anti-Oct-3/4 (sc-8628, Santa Cruz Biotechnology), mouse anti-α-tubulin (T5168, Sigma-Aldrich), mouse anti-Flag (F1804, Sigma-Aldrich), rabbit anti-Nup107 (raised against mNup107-His, amino acids 600–926), rabbit anti-Tuj1 (neuron-specific class III β-Tubulin; PRB-435P, BioLegend), mouse anti-Gapdh (GTX627408, Genetex), rabbit anti-Blbp (Fabp7; ab32423, Abcam), rat anti-Nestin (556309, BD pharmigen), mouse anti-Gata4 (sc-25310, Santa Cruz Biotechnology), rabbit anti-GFAP (Z0334, Dako), goat anti-Foxa2 (AF2400, R&D Systems), mouse anti-α-Sma (A5228, Sigma), rabbit anti-Nup98 (2292, Cell Signaling), rabbit anti-Sox2 (2748, Cell Signaling), mouse anti-V5 (46-0705, Life Technologies), mouse anti-Ring1b (39663, Active Motif), rabbit anti-Ring1b (5694S, Cell Signaling), rabbit anti-Rybp (ab5976, Abcam), rabbit anti-Cbx7 (ab21873, Abcam), rabbit anti-Nup50 (ab151567, Abcam), rabbit anti-Suz12 (ab12073, Abcam), rabbit anti-Lmnb2 (ARP46356; Aviva Systems Biology), rabbit anti-Mel-18 (sc-10744, Santa Cruz Biotechnology), and rabbit anti-Foxo1 (2880S, Cell Signaling).
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