Antibodies used in this study were anti-DNMT1 (Abcam), anti-5mC (Cell Signaling), anti-GAPDH, anti-ERK, anti-phospho-ERK (Cell signaling), anti-SMA (Sigma), anti-CD8 (Biolegend, BioXcell, or eBiosciences), anti-CD11c, anti-CD45, anti-F480, anti-MECA-79, anti-Vcam1, and anti-CD4 (all from BD Biosciences), anti-granzyme B (Cell Signaling), anti-mouse IgG (BioXcell), anti-PD-1 (CD279, BioXcell), and anti-PD-L1 (Genentech, MTA program). MGECs were isolated and cultured as described previously by our lab60 ,61 (link). Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza and cultured in EBM-2 supplemented with the EGM-2 bullet kit. Culture dishes were precoated with 1% gelatin. For the labeling of CD8+ T-cells, CellTrackerTM green dye was used (Molecular Probes). The culture of EO771 and PyMT cells was described previously62 (link),63 (link). Other reagents include a JAK2 inhibitor (AG490), NFκΒ inhibitor (JSH23, Sigma), TNFα (Peprotech), IFNγ (EMD Millipore), GSK3484862 (Med Chem Express), and 5-Azacytidine (Sigma).
Anti 5 mc
Manufactured by Cell Signaling Technology
Anti-5-mC is a laboratory product that detects and binds to 5-methylcytosine, a modified form of the DNA base cytosine. It can be used to study DNA methylation, a biological process involved in gene regulation and chromatin structure.
Automatically generated - may contain errors
Lab products found in correlation
Axioskop 2 plus, by Zeiss (4 mentions)
Anti lysozyme c, by Santa Cruz Biotechnology (2 mentions)
Ultracruz mounting medium with dapi, by Santa Cruz Biotechnology (2 mentions)
Apotome 2, by Zeiss (2 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Tissue tek oct compound, by Sakura Finetek (2 mentions)
Anti cd11c, by Thermo Fisher Scientific (1 mentions)
Anti sma, by Merck Group (1 mentions)
Anti granzyme b, by Cell Signaling Technology (1 mentions)
Anti pd l1, by Genentech (1 mentions)
Gsk 3484862, by MedChemExpress (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti dnmt1, by Abcam (1 mentions)
Anti phospho erk, by Cell Signaling Technology (1 mentions)
Anti f4 80, by BD (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
5 azacytidine, by Merck Group (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Egm 2 bulletkit, by Lonza (1 mentions)
5 protocols using anti 5 mc
1
Immunomodulatory Protocols for Cancer Research
2
Immunofluorescence Detection of 5-mC
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Treated cells were seeded on coverslips (Sarstedt Inc. TC coverslip 13 mm ST/CS200, Fisher Scientific) were fixed with 4% PFA in PBS for 15 min at room temperature. Cells were blocked with 1% BSA dissolved in PBS for 1 h after permeabilized with 0.1% Triton X-100 in PBS for 7 min, then incubated with primary antibody anti-5-mC (28692, Cell Signaling Technology) overnight at 4 °C, followed by Fluor 488-conjugated (1:200) incubation for 1 h at room temperature. The tablets were sealed with an anti-fluorescence quencher containing DAPI (P0131, Beyotime). IF images were acquired with a laser scanning confocal microscope (Andor) equipped with Imairs Viewer software.
3
DNA Methylation Analysis by 5mC/5hmC IP
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Two micrograms of sonicated DNA was denatured at 95 °C for 10 min, immediately cooled on ice for 10 min, and diluted in 500 μl of IP buffer (10 mM Na-Phosphate pH 7.0 140 mM NaCl 0.05% Triton X-100), 50 μl as input. Then, add 10 μl of anti-5mC (#28692; Cell Signaling Technology) or anti-5hmC (#39769; active motif; Cell Signaling Technology), IgG (#2729, Cell Signaling Technology) as control. After 2 h incubation at 4 °C with overhead shaking, 30 μl of 50% protein A/G-Sepharose magnetic beads were added and incubated for another 2 h. Put the tube on the magnetic beam for 5 min and discard supernatant. After five washes with IP buffer, DNA was eluted with Proteinase K for 2 h, then purified using the QIAQuick PCR Purification Kit (Qiagen) according to the manufacturer’s instructions. DNA was analyzed by quantitative real-time PCR by using a SYBR GreenER kit (Invitrogen). Primers sequences are provided in Table 1 . Fold enrichment was calculated as follows: %Input = 10% × 2^(CTinput−CTsample).
4
Immunohistochemical Analysis of Tissue and Organoid Samples
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For light microscopy, tissues and organoids were xed for 16 h in 10% neutral buffered formalin at 4 °C.
The tissues were embedded in para n and cut into 5-μm-thick sections. Sections were stained with hematoxylin and eosin (H&E) or subjected to immunohistochemistry. Organoids were embedded in Tissue-Tek ® O.C.T. Compound (Sakura Finetek), frozen at -20 °C, and cut into 10-µm-thick sections. For immunohistochemistry, para n sections were prepared by boiling in 10 mM citrate buffer pH 6.0 for 15 min. After blocking with 1% bovine serum albumin, sections were incubated for 16 h at 4 °C with the following primary antibodies: anti-lysozyme C (1:200, goat polyclonal, Santa Cruz Biotechnology); antiproliferating cell nuclear antigen (PCNA, 1:100, mouse monoclonal, Dako); anti-5-mC (1:300, rabbit monoclonal, Cell Signaling Technology); and anti-H3K4, 9, 27, 36, and 79me3 (1:300, rabbit monoclonal, Cell Signaling Technology). Before incubation with secondary antibodies, the tissues were washed to remove the unbound antibodies. The sections were then incubated with secondary antibodies (1:1000 dilution; Alexa Fluor 488 and/or Alexa Fluor 555, Invitrogen) for 1 h at 25 °C and mounted using UltraCruz™ Mounting Medium with DAPI (Santa Cruz Biotechnology). The samples were observed under a uorescence microscope (Axioskop 2 plus or Apotome. 2, Carl Zeiss).
The tissues were embedded in para n and cut into 5-μm-thick sections. Sections were stained with hematoxylin and eosin (H&E) or subjected to immunohistochemistry. Organoids were embedded in Tissue-Tek ® O.C.T. Compound (Sakura Finetek), frozen at -20 °C, and cut into 10-µm-thick sections. For immunohistochemistry, para n sections were prepared by boiling in 10 mM citrate buffer pH 6.0 for 15 min. After blocking with 1% bovine serum albumin, sections were incubated for 16 h at 4 °C with the following primary antibodies: anti-lysozyme C (1:200, goat polyclonal, Santa Cruz Biotechnology); antiproliferating cell nuclear antigen (PCNA, 1:100, mouse monoclonal, Dako); anti-5-mC (1:300, rabbit monoclonal, Cell Signaling Technology); and anti-H3K4, 9, 27, 36, and 79me3 (1:300, rabbit monoclonal, Cell Signaling Technology). Before incubation with secondary antibodies, the tissues were washed to remove the unbound antibodies. The sections were then incubated with secondary antibodies (1:1000 dilution; Alexa Fluor 488 and/or Alexa Fluor 555, Invitrogen) for 1 h at 25 °C and mounted using UltraCruz™ Mounting Medium with DAPI (Santa Cruz Biotechnology). The samples were observed under a uorescence microscope (Axioskop 2 plus or Apotome. 2, Carl Zeiss).
5
Immunohistochemical Analysis of Tissue and Organoid Samples
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+ Expand
For light microscopy, tissues and organoids were xed for 16 h in 10% neutral buffered formalin at 4 °C.
The tissues were embedded in para n and cut into 5-μm-thick sections. Sections were stained with hematoxylin and eosin (H&E) or subjected to immunohistochemistry. Organoids were embedded in Tissue-Tek ® O.C.T. Compound (Sakura Finetek), frozen at -20 °C, and cut into 10-µm-thick sections. For immunohistochemistry, para n sections were prepared by boiling in 10 mM citrate buffer pH 6.0 for 15 min. After blocking with 1% bovine serum albumin, sections were incubated for 16 h at 4 °C with the following primary antibodies: anti-lysozyme C (1:200, goat polyclonal, Santa Cruz Biotechnology); antiproliferating cell nuclear antigen (PCNA, 1:100, mouse monoclonal, Dako); anti-5-mC (1:300, rabbit monoclonal, Cell Signaling Technology); and anti-H3K4, 9, 27, 36, and 79me3 (1:300, rabbit monoclonal, Cell Signaling Technology). Before incubation with secondary antibodies, the tissues were washed to remove the unbound antibodies. The sections were then incubated with secondary antibodies (1:1000 dilution; Alexa Fluor 488 and/or Alexa Fluor 555, Invitrogen) for 1 h at 25 °C and mounted using UltraCruz™ Mounting Medium with DAPI (Santa Cruz Biotechnology). The samples were observed under a uorescence microscope (Axioskop 2 plus or Apotome. 2, Carl Zeiss).
The tissues were embedded in para n and cut into 5-μm-thick sections. Sections were stained with hematoxylin and eosin (H&E) or subjected to immunohistochemistry. Organoids were embedded in Tissue-Tek ® O.C.T. Compound (Sakura Finetek), frozen at -20 °C, and cut into 10-µm-thick sections. For immunohistochemistry, para n sections were prepared by boiling in 10 mM citrate buffer pH 6.0 for 15 min. After blocking with 1% bovine serum albumin, sections were incubated for 16 h at 4 °C with the following primary antibodies: anti-lysozyme C (1:200, goat polyclonal, Santa Cruz Biotechnology); antiproliferating cell nuclear antigen (PCNA, 1:100, mouse monoclonal, Dako); anti-5-mC (1:300, rabbit monoclonal, Cell Signaling Technology); and anti-H3K4, 9, 27, 36, and 79me3 (1:300, rabbit monoclonal, Cell Signaling Technology). Before incubation with secondary antibodies, the tissues were washed to remove the unbound antibodies. The sections were then incubated with secondary antibodies (1:1000 dilution; Alexa Fluor 488 and/or Alexa Fluor 555, Invitrogen) for 1 h at 25 °C and mounted using UltraCruz™ Mounting Medium with DAPI (Santa Cruz Biotechnology). The samples were observed under a uorescence microscope (Axioskop 2 plus or Apotome. 2, Carl Zeiss).
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