Multicolor immunofluorescence analyses were performed using 3-μm-thick sections of formalin-fixed paraffin-embedded (FFPE) tissues. Briefly, slides were deparaffinized in xylene and hydrated in a series of decreasing graded ethanol series. After heat-induced antigen retrieval in citrate buffer (pH = 6), samples were permeabilized with 0.5% Triton X-100, blocked with 5% goat serum-phosphate-buffered saline (PBS), and sequentially co-stained with antibodies recognizing VCAM-1 (Abcam, ab134047), CD248 (Abcam, ab217535), 4-HNE (Abcam, ab46545), FAPα (Invitrogen, BMS168; Abcam, ab218164), Mfap4 (Thermo, PA5-24865), Sparcl1 (Santa Cruz, sc-514275), F4/80 (Cell Signaling Technology, #70076), rabbit IgG Isotype Control (Invitrogen, 31235), mouse IgG Isotype Control (Invitrogen, 14-4714-82), and DAPI. A TSA indirect kit (PerkinElmer) was used according to the manufacturer’s instructions. Vectra® Polaris™ Imaging System (Akoya Biosciences) was used to collect images, and image analysis was performed using HALO™ Image Analysis Software.
Mouse igg isotype control
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Mouse IgG isotype control is a laboratory reagent used as a negative control in immunoassays. It is a non-specific mouse immunoglobulin G (IgG) antibody that does not bind to any known antigen. This control is used to establish baseline signals and determine non-specific binding in experiments involving mouse-derived antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit igg isotype control, by Thermo Fisher Scientific (4 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (3 mentions)
F4 80, by Cell Signaling Technology (2 mentions)
Tsa indirect kit, by PerkinElmer (2 mentions)
Proteinase k, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ab134047, by Abcam (1 mentions)
Vectra polaris imaging system, by Akoya Biosciences (1 mentions)
Ab46545, by Thermo Fisher Scientific (1 mentions)
Streptavidin peroxidase kit, by ZSGB-BIO (1 mentions)
P nfκb p65, by Thermo Fisher Scientific (1 mentions)
Anti mouse rabbit hrp dab cell tissue staining kit, by R&D Systems (1 mentions)
Flag m2 mouse monoclonal antibody, by Merck Group (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Bio-Rad (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Amersham imager 680, by Cytiva (1 mentions)
Qiaquick pcr kit, by Qiagen (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
28 protocols using mouse igg isotype control
1
Multicolor Immunofluorescence of FFPE Tissues
2
Immunohistochemical Analysis of Oxidative Stress
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Immunohistochemical staining was performed using a streptavidin–peroxidase kit (ZSGB-Bio, China). The primary antibodies targeted the following proteins or modifications: 8-OHdG (Abcam, ab48508), 4-HNE (Abcam, ab48506, ab46545), FAPα (Abcam, ab53066), GPX4 (Abcam, ab125066), GCLC (Abcam, ab53179), GCLM (Abcam, ab126704), p-NF-κB p65 (Thermo Fisher, 44-711G), rabbit IgG Isotype Control (Invitrogen, 31235), and mouse IgG Isotype Control (Invitrogen, 14-4714-82) with a standard avidin-biotin HRP detection system according to the instructions of the manufacturer (anti-mouse/rabbit HRP-DAB Cell & Tissue Staining Kit, R&D Systems, Minneapolis, MN). Tissues were counterstained with hematoxylin, dehydrated, and mounted. Quantitative analysis was performed with Image-Pro Plus Version 6.0 Software or HALO™ Image Analysis Software. For quantitative analysis using Image-Pro Plus, the percentage of positive cells was graded on a scale of 0 to 4 (0: negative, 1: 0–25%, 2: 26–50%, 3: 51–75%, 4: 76–100%). The staining intensity was further quantified from 0 to 3 (1: weak, 2: moderate, 3: intensive). The final score was obtained by multiplying the staining intensity score and percentage score.
3
EPRS-FLAG Immunoprecipitation and Interactome Analysis
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For assessing mutant EPRS association with MSC scaffold proteins, 1.5 × 106 of HEK293T shEPRS-inducible stable cells were seeded on 10 cm dishes and shRNA expression was induced with doxycycline the following day. Cells were transfected with 10 μg of pcDNA3 EPRS-FLAG (WT or mutant), using the PEI method (63 (link)) 24 h post doxycycline treatment. Cells were harvested 48 h post transfection and lysed in cell lysis buffer (Cell Signaling Technology) supplemented with protease inhibitor cocktail (Sigma-Aldrich).
For immunoprecipitation studies, 5 μg FLAG M2 mouse monoclonal antibody (Sigma-Aldrich F1804) or mouse IgG isotype control (Invitrogen 10400C) was conjugated to 25 μl Dynabeads protein G (Invitrogen) by incubating in 200 μl phosphate buffered saline with 0.01% Tween-20 (0.01% PBS-T) at room temperature for 10 min. Immunoprecipitation was performed by incubating 250 μl lysates (∼1200 μg protein) with antibody-conjugated beads overnight at 4 °C. Beads were washed three times with 0.02% PBS-T and boiled in SDS-PAGE loading buffer, followed by immunoblotting with the following antibodies: EPRS (Novus Biologicals NBP1-84929), AIMP2 (Thermo Scientific PA5-31306), AIMP3 (Invitrogen PA5-28283), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Bio-Rad). Immunoblots were developed for chemiluminescence and were imaged on an Amersham Imager 680 (Cytiva).
For immunoprecipitation studies, 5 μg FLAG M2 mouse monoclonal antibody (Sigma-Aldrich F1804) or mouse IgG isotype control (Invitrogen 10400C) was conjugated to 25 μl Dynabeads protein G (Invitrogen) by incubating in 200 μl phosphate buffered saline with 0.01% Tween-20 (0.01% PBS-T) at room temperature for 10 min. Immunoprecipitation was performed by incubating 250 μl lysates (∼1200 μg protein) with antibody-conjugated beads overnight at 4 °C. Beads were washed three times with 0.02% PBS-T and boiled in SDS-PAGE loading buffer, followed by immunoblotting with the following antibodies: EPRS (Novus Biologicals NBP1-84929), AIMP2 (Thermo Scientific PA5-31306), AIMP3 (Invitrogen PA5-28283), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Bio-Rad). Immunoblots were developed for chemiluminescence and were imaged on an Amersham Imager 680 (Cytiva).
4
ChIP Assay with Protein G Beads
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To perform ChIP assays, 1% formaldehyde for 10 min was used to cross-link the proteins to the DNA, then the reaction was quenched with 0.125 M glycine. After nuclei lysis, the lysates were sonicated and the immunoprecipitation was carried out using Dynabeads Protein G (Invitrogen, cat. 10004D). 0,1 mg/mL RNase A (Thermo Scientific) and Proteinase K (20 mg/mL, Thermo Scientific) were used to reverse the cross-links. The DNA was purified by QIAquick PCR kit (QIAGEN). The DNA levels were measured by real-time quantitative PCR. ChIP was performed with the following antibodies: anti-p53 (Leica, cat. P53-CM5P-L), and mouse IgG Isotype control (Invitrogen, cat. 10500C).
5
Aurora B Immunoprecipitation Protocol
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Cells were treated as described in Supplemental Fig. 4a and subject to immunoprecipitation using previously described methods following IP with Aurora B antibody (BD 611,082) or mouse IgG isotype control (Invitrogen 31903) (Swift et al. 2021a (link), b (link), c (link)).
6
Immunoprecipitation of CHAPSO-solubilized HeLa Membranes
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CHAPSO solubilized HeLa cell membranes (400 μg of protein/sample) were immunoprecipitated by anti-PS1-NTF or anti-IFITM3 antibody in immunoprecipitation buffer (20 mM Hepes (pH 7.5), 50 mM KCl, 2 mM EGTA, 0.25% CHAPSO and protease inhibitor mixture)6 (link),48 . In brief, protein A/G magnetic beads (Pierce) were washed then added to samples for 30 min at 4 °C with rotation as a pre-clear step. The supernatant from samples were incubated with 5 μg of purified PS1-NTF antibody, IFITM3 antibody, monoclonal IFITM3 antibody (9D11, generated in our lab), ImmunoPure rabbit IgG (Pierce) or mouse IgG isotype control (Invitrogen) for overnight at 4 °C with rotation. Beads were added to samples for 1 hr at RT with rotation. Beads were washed three times with 1 ml of immunoprecipitation buffer containing 1% CHAPSO and the beads were eluted in 30 μl of SDS-PAGE sample buffer with DTT for 10 min at RT. The eluted samples were subjected to SDS-PAGE and Western blotting (for source gels see Supplementary Figure 1 ).
7
Immunoprecipitation of CHAPSO-solubilized HeLa Membranes
8
Inducible Regulatory T Cell Differentiation
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Mouse naive CD4+ T cells (2 × 105 cells/well) were differentiated under suboptimal iTreg-skewing condition (TGF-β1 0.5 ng ml−1 and IL-2 20 ng ml−1) in each 96 flat-bottom well coated with 5 μg ml−1 of mouse IgG isotype control (Invitrogen), 5 μg ml−1 or 10 μg ml−1 of 6F01 along with 1 μg ml−1 of anti-CD3e antibody. In all, 1 μg ml−1 of anti-CD28 antibody was added into the culture medium. After 2 or 3 days of differentiation, the cells were harvested and were fixed/permeabilized using FIX & PERM™ Cell Permeabilization Kit (Thermo Fisher Scientific). The fixed cells were stained with anti-CD4-BV421 or APC, anti-Foxp3-FITC or APC, anti-p-Smad2/3-PE, anti-AKT antibody (Cell Signaling Technology), anti-p-AKT-APC, anti-mTOR antibody (Cell Signaling Technology), anti-p-mTOR-PE-cy7, anti-FoxO1 antibody (Cell Signaling Technology), and anti-p-FoxO1 antibody (Cell Signaling Technology). Anti-Rabbit IgG-PE secondary antibody (Invitrogen) was used for unconjugated antibodies. The stained cells were measured by Flow cytometry. iTreg cells differentiated under optimal iTreg-skewing condition (TGF-β1 5 ng ml−1 and IL-2 20 ng ml−1) were used as a positive control.
9
MUC1-Mediated Antibody-Dependent Cell Cytotoxicity
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HEK293WT control or membrane MUC1 reporter expressing cells were treated with 10 µM NSC80997 or DMSO for 4 h at 37 °C, washed with medium and re-seeded into 96-well flat bottom plates at a density of 5 × 105 cells in RPMI 1640 medium supplemented with 10% FBS and 1 × 2-mercaptoethanol (Gibco). Cells were allowed to adhere for 2 h and isotype, 1E3, 5E5, 6C5 or Mouse IgG isotype control (Invitrogen) mAbs (>2 µg/mL) were added 20 min prior to addition of peripheral blood lymphocytes (PBLs). Frozen PBLs from healthy donors were thawed and labeled with 5 µM CFSE (Invitrogen) according to the manufacturer’s instructions and added at 5:1, 10:1, and 20:1 ratios to the HEK293 cells. After overnight incubation, all cells were harvested and labeled with viability dye and analyzed by flow cytometry. Percentage lysis was calculated normalized to respective isotype controls.
10
Multiparametric Immunohistochemical Profiling
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In brief, slides were dewaxed, followed by antigen retrieval in citrate (pH = 6) or Tris-EDTA (pH = 9) buffer. After blocked with 5% goat serum, the slide was sequentially incubated with primary antibodies recognizing α-SMA (Abcam, ab7817), CD3 (Abcam, ab5690), CD4 (Abcam, ab183685), CD8 (Boster Bio, a02236-1), CD19 (Abcam, ab245235), Ly6G (Abcam, ab238132), NCR1 (Abcam, ab233558), F4/80 (Cell Signaling Technology, 70076 S), TGF-β (Abcam, ab215715), Sftpc (Abcam, ab90716), rabbit anti-mouse CD147 (Abcam, ab188190), mouse anti-human CD147 (prepared in our own lab), FSP1 (Proteintech, 20886-1-AP), rabbit IgG Isotype Control (Invitrogen, 31235), mouse IgG Isotype Control (Invitrogen, 14-4714-82). Goat anti-Mouse F(ab) fragment was used for mouse-on-mouse blocking. TSA-Indirect Kit (PerkinElmer, USA) was used according to the manufacturer’s manual. Images were analyzed with HALO™ Image Analysis Software or ImageJ Software.
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