The immunofluorescence staining was conducted as previously reported [55 (link)]. Detailed information for the antibodies are : TET1 (1:300, GeneTex, California, USA); 5hmC (1:500; Active Motif, California, USA); OCT-4 (1:300; Chemicon, Massachusetts, USA); stage-specific embryonic antigen 1 (SSEA-1; 1:200; Chemicon, Massachusetts, USA); H3K9me2 (1:500; Sino Biological Inc., Beijing, China); H3K27me3 (1:500; Sino Biological Inc., Beijing, China); Sox2 (1:200; Chemicon, Massachusetts, USA); proliferating cell nuclear antigen (PCNA; 1:200; Millipore, Massachusetts, USA); 5-bromo-2′-deoxyuridine (BrdU; 1:300; Santa Cruz, California, USA), FITC-conjugated secondary antibody (1:500; Chemicon, Massachusetts, USA), HOECHST33342 (Sigma-Aldrich, Missouri, USA). The immunofluoresence intensity was analyzed by ImageJ software (National Institutes of Health, USA).
H3k27me3
Manufactured by Sino Biological
Sourced in China
H3K27me3 is a histone modification that plays a role in epigenetic gene regulation. It is a post-translational modification where a methyl group is added to the 27th lysine residue of histone H3. This modification is associated with transcriptional repression and heterochromatin formation.
Automatically generated - may contain errors
Lab products found in correlation
H3k9me2, by Sino Biological (2 mentions)
Hdac1, by Sangon (2 mentions)
Fitc conjugated secondary antibody, by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Pgp 9, by Bioss Antibodies (1 mentions)
Glut2, by Bioss Antibodies (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Horseradish peroxidase conjugated anti rabbit antibody, by Beyotime (1 mentions)
Proliferating cell nuclear antigen (pcna), by Boster Bio (1 mentions)
Jmjd3, by Abcam (1 mentions)
β actin, by Sino Biological (1 mentions)
Bca protein quantification kit, by Vazyme (1 mentions)
4 protocols using h3k27me3
1
Immunofluorescence Staining Protocol for Stem Cell Markers
2
Immunofluorescent Staining Protocol for Tissue and Cell Samples
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For slides of tissues, before fixation, they were firstly dewaxed and soaked in boiling citrate buffer for 15 to 25 min to get natural cooling. For the cell samples, they were directly fixed in 4% PFA in PBS for 15 min and 0.5% Triton X-100 in PBS for 10 min, blocked in 4% goat serum at room temperature for 30 min, and then exposed to primary antibody TET1 (1:500; Cell Signaling Tecnology, Inc., USA), GLUT2 (1:200; Bioss, Beijing, China), PDX1 (1:200; Bioss, Beijing, China), PGP9.5 (1:50; Bioss, Beijing, China), VASA (1:100; Sangon Biotech, Shanghai, China), CCND1 (1:100; BOSTER, Wuhan, China), KI67 (1:100; Bioss, Beijing, China), HDAC1 (1:100; Sangon Biotech, Shanghai, China) and H3K27me3 (1:200; Sino Biological Inc.) overnight at 4 °C. After three 5 min washes in PBS, samples were incubated with secondary Alexa 594 anti-rabbit/mouse IgG antibody (1:200; Invitrogen) at room temperature for 30 min, followed by three washes in PBS. Cell nuclei were stained using DAPI. Images were captured using a Leica fluorescence microscope (Hicksville, NY, USA)43 (link).
3
Western Blot Analysis Procedure
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Western blot analysis procedure was described previously [55 (link)]. The primary antibodies were listed as follows: GAPDH (1:3000, Genesci, Shanghai, China); TET1 (1:500, GeneTex, California, USA); H3K9me2 (1:1000; Sino Biological Inc., Beijing, China), H3K27me3 (1:1000; Sino Biological Inc., Beijing, China), and OCT4 (1:500; Chemicon, Massachusetts, USA). Horse-radish peroxidase-conjugated anti-rabbit antibody (1:1000, Beyotime, Beijing, China) or anti-mouse antibody (1:2000, Beyotime, Beijing, China) was used as secondary antibodies.
4
Quantification and Western Blot Analysis
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Proteins were extracted from stably transfected cells and then the protein concentration was detected using the BCA Protein Quantification Kit (Vazyme, Piscataway, NJ, USA). After heat denaturation in 5% SDS–PAGE sample loading buffer, the protein samples were resolved by SDS-PAGE and transferred to a PVDF membrane. The samples were probed with β-ACTIN (1:1000; Sino Biological Inc., Beijing, China), S6/pS6 (1:1000; Cell Signaling Tecnology, Inc., USA), EZH2 (1:500; Sangon Biotech, Shanghai, China), JMJD3 (1:800; Abcam Inc., USA), ERK/pERK (1:500; Cell Signaling Tecnology), PCNA (1:500; BOSTER, Wuhan, China), SIN3A (1:300; Bioss, Beijing, China), HDAC1 (1:500; Sangon Biotech, Shanghai, China), OCT4 (1:500; Santa Cruz, Inc., USA) and H3K27me3 (1:1000; Sino Biological Inc., Beijing, China) as previously described54 (link). The secondary antibody was horseradish peroxidase-conjugated anti-rabbit/mouse IgG (1:1,000, Bioworld, Nanjing, China). The detection was performed using Thermo Scientific Pierce ECL western blotting substrate, and the results were analyzed using a Tanon-410 automatic gel imaging system.
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