Immunohistochemical staining was performed as described [26 (link)]. Slides from nude mice were incubated overnight with primary antibodies against ki67 (#ab15580, Abcam), p21 (#ab15580, Abcam) at 4 °C. The complex was observed by DAB complex, and the nuclei were counterstained with haematoxylin. The immunoreactivity in each section was assessed by at least two experienced pathologists and scored by semi-quantitative H-score approach [27 (link)].
Ab15580
Manufactured by Abcam
Sourced in United Kingdom, United States, China, Germany, Japan, Netherlands
Ab15580 is a primary antibody that detects the protein NRIP1 (Nuclear Receptor Interacting Protein 1). It is a mouse monoclonal antibody that can be used in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab6721, by Abcam (44 mentions)
Ab28364, by Abcam (30 mentions)
Ab150077, by Abcam (26 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (26 mentions)
Ab205718, by Abcam (22 mentions)
Ab5694, by Abcam (21 mentions)
Ab92547, by Abcam (17 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (17 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (17 mentions)
Ab2302, by Abcam (12 mentions)
Ab16669, by Abcam (11 mentions)
A7811, by Merck Group (11 mentions)
Ab92552, by Abcam (9 mentions)
Ab205921, by Abcam (9 mentions)
Anti ki67, by Abcam (9 mentions)
Ab182422, by Abcam (9 mentions)
Triton x 100, by Merck Group (9 mentions)
Lsm 880, by Zeiss (9 mentions)
Ab97959, by Abcam (8 mentions)
Ab18197, by Abcam (8 mentions)
1 122 protocols using ab15580
1
Immunohistochemical Evaluation of Ki67 and p21
2
Immunohistochemistry and Immunofluorescence Analysis
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Paraffin-embedded liver Sects. (4 µm thick) were used for immunohistochemistry (IHC) experiments. The sections were dewaxed, rehydrated, and quenched with 3% H2O2, followed by heat-induced epitope retrieval in 10 mM citrate buffer (pH 6) at 95 °C for 20 min. Nonspecific antigens were blocked with 1% BSA (cat: A7906, Sigma-Aldrich). Anti-Ki67 (1:500, ab15580, Abcam) and anti- Hnf4α antibodies (1:500, 3113S, CST) were incubated overnight at 4 °C. Goat-anti-rabbit fluorescein isothiocyanate-labeled IgG or goat-anti-mouse rhodamine IgG (1:200, Proteintech, Rosemont, IL, USA) were incubated at 4 °C for 2 h, followed by 4′,6-diamidino-2-phenylindole (DAPI) staining (Abcam) the cell nucleus. Slides were mounted and visualized using an OLYMPUS microscope.
Primary hepatocytes or macrophages were seeded in a cell culture dish for immunofluorescence experiments. The cells were fixed and permeabilized at 4 °C for 30 min. After incubation with anti-Tet2 (1:100, ab124297, Abcam), anti-Ki67 (1:100, ab15580, Abcam), and anti-Stat1 antibodies (1:100, 9176, CST) at 4 °C overnight, the cells were washed with PBS and stained with goat-anti-rabbit FITC-labeled IgG or goat-anti-mouse rhodamine IgG (1:200, Proteintech) at 4 °C for 2 h, followed by DAPI staining (Abcam). The cells were viewed using a Zeiss Confocal Microscope Imaging System (Carl Zeiss, Jena).
Primary hepatocytes or macrophages were seeded in a cell culture dish for immunofluorescence experiments. The cells were fixed and permeabilized at 4 °C for 30 min. After incubation with anti-Tet2 (1:100, ab124297, Abcam), anti-Ki67 (1:100, ab15580, Abcam), and anti-Stat1 antibodies (1:100, 9176, CST) at 4 °C overnight, the cells were washed with PBS and stained with goat-anti-rabbit FITC-labeled IgG or goat-anti-mouse rhodamine IgG (1:200, Proteintech) at 4 °C for 2 h, followed by DAPI staining (Abcam). The cells were viewed using a Zeiss Confocal Microscope Imaging System (Carl Zeiss, Jena).
3
Xenograft Tumor Model for Oncogene Study
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Approval for the animal experiments was obtained from the Animal Care and Use Committee of The Central Hospital of Enshi Tujia and Miao Autonomous Prefecture and all animal procedures followed international guidelines. sh‐NC‐ or sh‐circ_0026134‐transduced H520 cells (5 × 106 cells) were subcutaneously injected into male BALB/c mice aged 6 weeks (Vital River Laboratory; n = 6 per group). The developing tumors were periodically measured with a digital caliper and tumor volume was determined as follows: volume = length × width2 × 0.5. On day 40 after cell injection, the mice were sacrificed and the xenograft tumors were excised for weight measurement and gene expression analysis by qRT‐PCR and western blot. Immunohistochemistry analysis for Ki‐67 level measurement in the xenograft tumors was done as described.20 Briefly, the tissue paraffin sections were incubated with Ki‐67 antibody (1:100; ab15580, Abcam) and biotinylated anti‐rabbit secondary antibody (1:1000; ab15580, Abcam), followed by the incubation with the DAB detection kit as described by the manufacturers (Abcam).
4
Immunofluorescence Analysis of PCa Cells
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Cells were fixed and permeabilized with Perm/Wash Buffer (Cat. # 554723, BD Biosciences). Samples were blocked with Image-iT FX signal enhancer for 30 min and incubated for 2 hours at room temperature with primary antibodies combined with reagents of Zenon Alexa Fluor 488 (green) or 555 (red) labeling kit (Invitrogen). To detect Ki-67 in PCa cells co-cultured with MC3T3-E1 cells in vitro, human Ki-67 (cat. # ab15580, Abcam) and HLA-ABC (Cat. # 311402, BioLegend) antibodies were used as primary antibodies. To detect Ki-67 in PCa cells in bone marrow sections, human Ki-67 (cat. # ab15580, Abcam) and pan cytokeratin (cat. # ab867364, Abcam) antibodies were used as the primary antibody. To detect human TGFBR2 and TGFBR3, human TGFBR2 (cat. # ab61213, Abcam) and human TGFBR3 (cat. # ab78421, Abcam) antibodies were used as the primary antibody. After washing with PBS, the slides were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen). Images were taken with Nikon A-1-B confocal microscope.
5
Immunostaining of Ki67 for Cell Proliferation
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Cells were fixed using 4% paraformaldehyde and subsequently permeabilized with 0.1% Triton X-100. After blocking with 1% BSA, cells were incubated overnight at 4 °C with a primary antibody (1:500 dilution) for cell proliferation Ki67 (ab15580, Abcam, Cambridge, MA). Cells were then incubated with secondary antibodies (1:1000 dilution, A-11034, Invitrogen, Carlsbad, CA, USA) and DAPI (Vector Laboratories, Burlington, ON, Canada) for nuclei at room temperature for 2 h. The image was captured using a Nikon TE-2000 digital microscope equipped with a Hamamatsu C4742-80-12AG camera. The number of antibody-labeled cells was quantified from five separate images by three blinded investigators.
Similarly, sections of terminal ileum were immunostained for Ki67 (ab15580, Abcam, Cambridge, MA) using the same protocol as above. For subsequent reactions, a streptavidin–biotin complex peroxidase kit (LASB + Kit, Dako, Denmark) was used and slides analyzed the same way as mentioned above.
Similarly, sections of terminal ileum were immunostained for Ki67 (ab15580, Abcam, Cambridge, MA) using the same protocol as above. For subsequent reactions, a streptavidin–biotin complex peroxidase kit (LASB + Kit, Dako, Denmark) was used and slides analyzed the same way as mentioned above.
6
Immunohistochemical and Immunofluorescent Analysis of Tumor Samples
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Tumor sections (4 μm) were prepared on slide glasses, and sectioned slides were deparaffinized in xylene and gradually hydrated using an alcohol gradient from 100 to 70%. After washing with PBS, slides were warmed in a microwave with citrate buffer following incubation in 3% H2O2. Sections were then incubated with 5% bovine serum albumin in PBS to block nonspecific binding. Ki67 (1:100, ab15580; Abcam, Cambridge, UK) and microtubule-associated protein 1A/1B-light chain 3 (LC3-II) (1:200, L7543; Sigma-Aldrich) antibodies were added to the slides and incubated overnight at 4 °C. Sections were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. After washing, the sections were incubated with DAB substrate. Finally, hematoxylin staining was performed after mounting the slides using Permount mounting medium (Sigma-Aldrich). Cell proliferation was detected also by immunofluorescence staining using an antibody against Ki67 (1:100, ab15580; Abcam) for 2 h. Next, tissue sections were incubated with Alexa Fluor-labeled secondary antibodies (1:500) for 20 min at 37 °C and washed with PBS. The coverslips were mounted in Prolong Gold anti-fade reagent with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) for 10 min at room temperature and examined by fluorescence microscopy (Olympus CKX41 and U-RFLT 50).
7
Immunohistochemical Profiling of Endothelial Cells
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The following chemicals, unless specifically indicated, were from Sigma-Aldrich (St. Louis, MO, United States): forskolin (F6886), epinephrine (E4642), heparin (H3149), 4', 6-diamidino-2 phenylindole dihydrochloride (DAPI, D8417), mitomycin C (M5353), and dimethylsulfoxide (D2650). Anti ERK2 (rabbit, 1:1000, sc-292838, RRID: AB_2650548 ; Santa Cruz Biotechnology, Dallas, TX, United States) and anti-zyxin (mouse, 1:500, H00007791-M01, RRID: AB_2221180 ; Abnova, Taipei, Taiwan) were used for western blot. Anti Pecam1 (rat, 1:500, 553370, RRID: AB_394816 ; BD Bioscience, San Jose, CA, United States) and anti Ki67 (rabbit, 1:500, ab15580, RRID: AB_443209 ; Abcam, Cambridge, MA, United States) were used for whole-mount staining. anti-zyxin (mouse, 1:500, H00007791-M01, RRID: AB_2221180 ; Abnova, Taipei, Taiwan) and anti Ki67 (rabbit, 1:500, ab15580, RRID: AB_443209 ; Abcam, Cambridge, United Kingdom) were used for immunohistochemistry and immunofluorescence staining.
8
Immunohistochemical and Immunocytochemical Analysis of Cell Cycle Markers
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Immunohistochemical staining for H2A histone family, member X (γ-H2AX, Abcam, ab81299), BrdU (Abcam, ab6326), Ki67 (Abcam, ab15580), Cyclin A2 (Abcam, ab181591), Cyclin E1 (Abcam, ab52189) and Cyclin D1 (Abcam, ab134175) were performed on 4% paraformaldehyde-fixed liver sections. 5μm-thick slices were incubated with primary antibodies at 4? overnight followed by biotinylated secondary antibody and the avidin/biotin horseradish peroxidase system (Vectastain DAB Kit; Vector Laboratories, Burlingame, CA). The nuclei were counterstained with hematoxylin (Sigma-Aldrich, St. Louis, MO) and the sections were covered in neutral balsam (Solarbio, Beijing, CHN). Immuno-fluorescent staining for β-catenin (Sigma-Aldrich, MAB2081) and PHH3 (Roche, 760-4591) were detected on paraffin-embedded liver section. Slides were incubated with primary antibodies at 4 °C overnight and then with secondary antibodies conjugated with fluorescent dye at 37°C for 30 min.
Immunocytochemical staining for BrdU (Abcam, ab6326) and Ki67(Abcam, ab15580), cells were fixed, permeabilized and blocked, then incubated with primary antibody, followed by fluorescence-tagged secondary antibodies. Nuclei were counterstained with Hoechst 33258 (Sigma-Aldrich, St. Louis, MO). Images were acquired with a 50i Nikon fluorescence microscope (Nikon, Melville, NY).
Immunocytochemical staining for BrdU (Abcam, ab6326) and Ki67(Abcam, ab15580), cells were fixed, permeabilized and blocked, then incubated with primary antibody, followed by fluorescence-tagged secondary antibodies. Nuclei were counterstained with Hoechst 33258 (Sigma-Aldrich, St. Louis, MO). Images were acquired with a 50i Nikon fluorescence microscope (Nikon, Melville, NY).
9
Quantifying Epidermal Cell Proliferation
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The RNAScope® Multiplex fluorescence V2 system (Advanced Cell Diagnostics, Inc., Newark, CA, USA) was applied to in situ hybridization according to the manufacturer's instructions. Briefly, 10 μm paraffin skin sections from the AEW and Water groups were deparaffinized, rehydrated, washed in diethylpyrocarbonate (DEPC) distilled water, and stained with the following probes: mouse-Col17a1 (552141-C1; Advanced Cell Diagnostics) and mouse-Cux1 (442931-C2; Advanced Cell Diagnostics). After completing RNAScope, the skin sections were immediately subjected to Ki67 (ab15580, 1:250; Abcam, Waltham, MA, USA) immunofluorescence staining. The fluorescence intensities of Col17a1 and Cux1 in proliferating basal cells (stained positive for anti-Ki67 antibody in the epidermis) were quantified in both the AEW and Water groups. For skin sections from patients with psoriasis, we performed double immunofluorescence staining with Ki67 (ab15580, 1:250; Abcam) and Cux1 (sc-514008, 1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).
10
Immunohistochemistry for Ki67 in Paraffin Sections
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Immunohistochemistry was performed on 4-μm-thick sections of paraffin-embedded specimens using anti-Ki67 (ab15580, abcam) at 4 degrees in a moist chamber overnight. Then treated with the biotin-labelled secondary antibody, a VECTASTAIN Elite ABC peroxidase kit (PL-6100, Vector laboratories), followed by colour development with DAB. 40× images were acquired with Ni-E brightfield microscope (Nikon, Tokyo, Japan)
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