Flag-RECS1, MYC-RECS1, BAX, ERp57, LAMP-1/2, LGALS1/galectin-1, and LGALS3/galectin-3 proteins were visualized by immunofluorescence. The following antibodies were used: anti-Flag (F7425, Sigma-Aldrich), anti-Flag M2 (F1804, Sigma-Aldrich), anti-TBMIM1 (MBS1499661, MyBioSource), anti-ERp57 (ab13506, Abcam), anti–LAMP-1 (an24170, Abcam), LAMP-2 (ab18528, Abcam), BAX (ab5714, Abcam), anti-LGAL1/galectin-1 (ab25138, Abcam), and LGAL3/galectin-3 (556904, BD Biosciences). All antibodies were diluted 1:1000. We used a sensitive method based on a confined displacement analysis algorithm to calculate colocalization coefficients between Flag-RECS1 and ERp57 (ER), GM130 (Golgi apparatus), and LAMP-1/2 (endosomes/lysosomes) (63 (link)). The colocalization of images was performed as previously described (63 (link)).
Ab25138
Manufactured by Abcam
Ab25138 is a lab equipment product manufactured by Abcam. It is a core functional component designed for use in scientific research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab199428, by Abcam (2 mentions)
Ab155244, by Abcam (2 mentions)
Ab124762, by Cell Signaling Technology (2 mentions)
Ab218107, by Abcam (2 mentions)
Ab25455, by Thermo Fisher Scientific (2 mentions)
Ab15580, by Abcam (2 mentions)
Axiovert microscope, by Zeiss (2 mentions)
Sc 376373, by Abcam (2 mentions)
Ma1 19063, by Agilent Technologies (2 mentions)
M7018, by Abcam (2 mentions)
Ab5714, by Abcam (1 mentions)
Ab13506, by Abcam (1 mentions)
Ab18528, by Abcam (1 mentions)
Mbs1499661, by MyBioSource (1 mentions)
Anti flag m2 f1804, by Merck Group (1 mentions)
Ab14592, by Abcam (1 mentions)
Cytokines, by Thermo Fisher Scientific (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab51502, by Abcam (1 mentions)
Ab5176, by Abcam (1 mentions)
8 protocols using ab25138
1
Subcellular Localization of Key Proteins
2
Cytokine-Responsive Protein Regulation
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Cytokines were obtained from Peprotech (Rocky Hill, NJ). S-Nitrosocysteine (CysNO) was synthesized by combining an equimolar concentration of L-cysteine with sodium nitrite in 0.2 N HCl and used within 1 h. We obtained antibodies against the following: galectin 1 (ab25138), NEDD4 (ab14592) and serpin B6 (ab97330) from Abcam (Cambridge, MA) (respective catalog numbers in parentheses). Tissue culture media and reagents were from Biological Industries (Beit Haemek, Israel). His-tagged proteins Trx(C35S) was expressed and purified from Escherichia coli as previously described [16 (link)]. Other materials were obtained from Sigma unless otherwise indicated.
3
Placental Gal-1 Protein Expression
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Placental protein was extracted by radio-immunoprecipitation assay (RIPA), and 1 ml tissue lysate and 10 μl phenylmethylsulfonyl fluoride (PMSF) were added to each 100 mg placenta tissue. The tissues were subsequently homogenized on ice and lysed for 5 min three times. The protein concentration was determined using bicinchoninic acid (BCA). The proteins to be tested were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane, followed by 1 h blocking at room temperature (RT) in 7% non-fat milk of PBS with 0.07% Tween-20. Rabbit polyclonal antibody Gal-1 (Abcam, ab25138) was added to the blots and incubated overnight at 4 °C. Following this, the membrane was washed with tris-buffered saline (TBS) and Tween 20, goat anti-rabbit secondary antibody (Abcam, ab205718) was added, and the membrane was placed in room temperature conditions and incubated for 2 h. The membrane was then washed with TBS and exposed with enhanced chemiluminescence (ECL). GAPDH was used as an internal loading control. The band intensity was quantified using Image J software.
4
Comprehensive Antibody Panel for Neural Cell Characterization
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Antibodies for CTIP2 (ab18465, 1:500), CUX1 (ab54583, 1:500), PE-conjugated anti EGFR (ab231, 1:50), LGALS1 (ab25138, 1:1,000), Lin28 (ab46020, 1:1,000), PHH3 (ab5176, 1:250), PLZF (ab104854, 1:100), POU3F2 (ab94977, 1:1,000), SATB2 (ab51502, 1:50), SOX1 (1:1,000), SOX2 (ab79351, 1:500), TBR1 (ab31940, 1:200), TBR2 (ab23345, 1:200) were from Abcam. Antibodies for BrdU (347580, 50 μl per test), KI67 (556003, 1:1,000), phycoerythrin-conjugated (PE) SSEA-3 (560237, 20 μl per test), PE-conjugated F11R (552556; 20 μl per test), Alexa Fluor 647-conjugated TRA-1-60-647 (560850, 5 μl per test), Alexa Fluor 647-conjugated TUJ1 (560340, 1:500) were from BD Biosciences. Antibodies for DCX (AB2253, 1:5,000), O4 (MAB345, 1:25), RELN (MAB5364, 1:200), Tyrosine Hydroxylase (TH, AB152, 1:500) were purchased from Millipore. Antibodies for FABP7 (51010-1-AP, 1:100), S100B (15146-1-AP, 1:100) were from ProteinTech. Antibodies for AP2α (3B5 concentrated, 1:100) and PAX6 (supernatant, 1:16) were from DSHB. Antibody for NESTIN (MO15012, 1:500) was from Neuromics. Antibody for GFAP (Z0334, 1:2,000) was from DAKO. Antibody for β-3-Tubulin (PRB-435P, 1:1,000) was from Covance. Antibody for GLAST (ACSA-1; 130-095-822, 1:10) was from Miltenyi Biotec. Antibody for OTX2 (AF1979; 1:40) was from R&D. Antibody for FOXA2 (SC-6554; 1:100) was from Santa Cruz Biotechnology.
5
Dual Immunofluorescence Staining Protocol
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Double immunofluorescence staining was done as described [3 (link)]. Human-specific anti-DEspR mAb (5g12e8) and human-specific anti-Galectin-1 antibody (Abcam, cat#ab25138) were labeled with AlexaFluor(AF)-488 or AF568, and used at 1 μg/ml for anti-Galectin-1 and at 100 μg/ml for 5g12e8 mAbs on fixed, paraffin-embedded sections following antigen-retrieval. Digital photomicroscopy was done using a Zeiss Axioskop fluorescence microscope with auto-exposure settings.
6
Galectin Immunohistochemistry in FFPE and Frozen Tissue
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FFPE tissue sections were de-paraffinized and rehydrated, followed by heat-induced epitope retrieval. Sections were then fixed in 4% PFA for 10 min at room temperature, washed in PBS for 5 min, permeabilized in 0.3% Triton X-100 for 3 min, then washed twice in PBS for 5 min before incubation with primary antibodies at 4°C overnight. The next day, sections were washed three times in PBS and mounted with ProLong Gold Antifade with DAPI (Thermo Fisher). For Gal-3 staining, frozen tissue sections were air-dried, incubated for 5 min in ice cold methanol/acetone (1:1), and incubated with primary antibody at 4°C overnight, then processed as FFPE tissue sections from hereon. The following primary antibodies were used: anti-galectin-1 (1:500, ab25138, Abcam), anti-galectin-3 (1:500, MAB11541, R&D), anti-galectin-8 (1:200, ab109519, Abcam), and anti-cytokeratin 10 (K10) (1:100, M7002, Dako (Agilent). The following secondary antibodies were used: goat anti-mouse Alexa Flour 594™, goat anti-rabbit Alexa Flour 488™, and goat anti-mouse Alexa Flour 488™. Confocal images were acquired with LSM 780 AxioObserver (Zeiss) with a 20x objective. Quantification was performed using Zen software, based on a trained intellesis segmentation model. Data is presented as the fold change of the average of triplicates.
7
Immunofluorescence Staining of Trophoblast Cells
8
Immunofluorescence Staining of Trophoblast Cells
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Cells plated on Matrigel-coated coverslips were fixed in 4% paraformaldehyde (PFA) for 15 min, permealized with 0.25% Triton-X for 10 min, blocked with 10% goat serum/0.1% Tween-20/PBS at RT for 1 h. Cells were incubated with primary antibodies diluted in 1% BSA/PBST at 4 °C overnight, washed with 0.1% Tween-20/PBS for 3 times, then incubated with secondary antibodies and DAPI for 1 h, washed with PBST, then mounted. Images were captured with a Zeiss Axiovert microscope and Axiovision imaging software SE64 V4.9.1. Primary antibodies used in this study were GATA3 (Abcam #ab199428), TFAP2C (Abcam #ab218107), TEAD4 (Abcam #ab155244), p63 (Abcam #ab124762), CDX2 (Cell Signaling Technology #12306), ELF5 (Santa Cruz Technology # SC-376373), HLA-G (clone 4H84 Abcam #ab25455), hCG (Invitrogen #MA1–19063) CK7 (DAKO #M7018), hPL (Abcam #ab25138), and Ki67 (Abcam #ab15580).
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