Paraffin-embedded biopsies and 3D organoid products were serially sectioned and subjected to hematoxylin and eosin (H&E) staining, immunofluorescence (IF), and immunohistochemistry (IHC) as described previously.8 (link), 30 (link), 31 (link) H&E-stained slides for 3D organoids and corresponding biopsies were evaluated by a pathologist (A.K.S.) blinded to molecular and clinical data. In brief, sections were incubated overnight at 4°C with anti-IVL (mouse monoclonal anti-IVL I9018, 1:100; Sigma-Aldrich) for IF and anti-Ki-67 (rabbit monoclonal anti-Ki-67 ab16667; 1:200; Abcam, Cambridge, UK) or anti-Notch1 (rabbit monoclonal anti-ICN1Val1744 D3B8, 1:200; Cell Signaling, Danvers, MA) for IHC. Stained objects were captured and imaged with a Nikon Microphot microscope with a digital camera. Ki-67 labeling index was determined by counting at least 6 organoids per group. Basaloid cell content analysis was determined as a percentage of the total height of basaloid cell layers per organoid diameter spanning both basaloid cell layers and the differentiated cell layers including keratinized inner cell mass in H&E-stained organoids with 3 areas measured per organoid in least 6 organoids per group.
Anti ki67 ab16667
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-Ki67 (ab16667) is a primary antibody that targets the Ki-67 protein, which is a well-established marker of cellular proliferation. It can be used in various immunoassay applications to detect the presence and distribution of Ki-67 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (2 mentions)
Anti phospho mek1 2, by Cell Signaling Technology (2 mentions)
Anti erk1 2, by ABclonal (2 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (2 mentions)
Anti mek1 2, by Cell Signaling Technology (2 mentions)
Microphot microscope, by Nikon (1 mentions)
Histofine, by Nichirei Biosciences (1 mentions)
Anti ps6, by Cell Signaling Technology (1 mentions)
Anti e cadherin ab1416, by Abcam (1 mentions)
Anti β actin sc 1616, by Santa Cruz Biotechnology (1 mentions)
Anti egfr sc 03, by Santa Cruz Biotechnology (1 mentions)
Anti mouse antibody, by Santa Cruz Biotechnology (1 mentions)
Ab92547, by Abcam (1 mentions)
Anti vimentin, by Abcam (1 mentions)
Mounting medium, by Thermo Fisher Scientific (1 mentions)
Mhs16, by Merck Group (1 mentions)
Ht110132, by Merck Group (1 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Crystal violet, by Merck Group (1 mentions)
17 protocols using anti ki67 ab16667
1
Histological and Immunohistochemical Analysis of 3D Organoids
2
Immunohistochemical Analysis of Kidney and Liver
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Sliced paraffin sections were immersed in xylene for deparaffinization, rehydrated to PBS- from ethanol, and then heated in a microwave oven to 100°C for 15 min in 10 mM sodium citrate buffer (pH 6.0) to unmask antigens. The sections were incubated in 0.3% H2O2/methanol to destroy endogenous peroxidase activity, and blocked with 1% bovine serum albumin and 0.05% NaN3 in PBS- for 30 min at room temperature, and then incubated with a primary antibody overnight at 4°C [23 (link), 24 (link)]. The primary antibodies used for immunohistochemistry were anti-pERK1/2 (M8159, dilution 1:200; Sigma), anti-pS6 (#4858, dilution 1:100; Cell Signaling), anti-Ki67 (ab16667, dilution 1:100; Abcam). The sections were rinsed and incubated with a biotinylated anti-mouse and anti-rabbit IgG/IgA/IgM secondary antibody (Histofine; Nichirei Biosciences, Tokyo, Japan) for immunohistochemical staining. Immune reaction products were developed with 3,3′-diaminobenzidine. Cells with positive signals were counted in 10 random fields of either kidney or liver sections obtained from 6 rats in each group by a naive observer using a 40× objective. In the kidney, we counted positive cells in both normal and cystic structural areas.
3
Antibody Validation for Cell Signaling
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The primary antibodies used in this study are listed as follows: anti-EGFR (sc-03; Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-Ki-67 (ab16667; Abcam, Cambridge, UK), anti-E-cadherin (ab1416; Abcam), antivi-mentin (ab92547; Abcam), anti-β-actin (sc-1616; Santa Cruz Biotechnology), and antigreen fluorescence protein (GFP; sc-8334; Santa Cruz Biotechnology). Antimouse antibodies (Santa Cruz Biotechnology) and antirabbit antibodies (Zhongshan Goldbridge Biotechnology, Beijing, China) were used for Western blot in this study.
4
Histological Analysis of Prostate Tissues
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Fixed prostate tissues were embedded in paraffin wax and cut into 5 μm thick sections. The sections were deparaffinized, rehydrated, and stained with Mayer's hematoxylin (MHS-16, Sigma-Aldrich) and eosin (HT110-1-32, Sigma-Aldrich) (H&E) solution using standard procedures. The sections were mounted with mounting medium (Invitrogen, Carlsbad, CA) and observed under light microscopy with bright-field illumination (Olympus, Tokyo, Japan).
Immunohistochemistry was performed using a Vectastain Elite ABC Kit (Vector Laboratories Inc., Burlingame, CA) according to the manufacturer's instructions. After antigen retrieval processing, the sections were blocked in normal serum and then incubated with anti-Ki-67 (ab16667, Abcam, Cambridge, UK) antibody overnight at 4°C. On the next day, the sections were incubated in biotinylated-secondary antibody and developed using a DAB peroxidase substrate kit (Vector Laboratories) until a signal was seen.
Immunohistochemistry was performed using a Vectastain Elite ABC Kit (Vector Laboratories Inc., Burlingame, CA) according to the manufacturer's instructions. After antigen retrieval processing, the sections were blocked in normal serum and then incubated with anti-Ki-67 (ab16667, Abcam, Cambridge, UK) antibody overnight at 4°C. On the next day, the sections were incubated in biotinylated-secondary antibody and developed using a DAB peroxidase substrate kit (Vector Laboratories) until a signal was seen.
5
Xenograft Tumor Establishment and Rb Silencing
6
Xenograft Tumor Establishment and Rb Silencing
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All animal experiments were approved by the University of Massachusetts Boston Institutional Animal Care and Use Committee and were conducted following institutional and national (USA) guidelines. To establish the xenograft tumors, C4–2-tet-shRB cells mixed with Matrigel (BD Biosciences) were subcutaneously injected (2×106 cells per injection) on flanks of castrated male SCID mice (~6-week old, Taconic). Xenograft tumors were further passaged in castrated mice. To silencing Rb, mice were fed with doxycycline supplemented food and drinking water. Tumor length (L) and width (W) were measured by caliper every other day during the treatment period and tumor volumes were calculated (L × W2/2). The tumor cell proliferation was examined by immunohistochemistry staining of Ki67 (anti-Ki67: ab16667 from Abcam, performed by iHisto).
7
Colorectal Cancer Pathogenesis Biomarkers
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Crystal violet, paraformaldehyde, fibronectin, laminin, poly L-lysine, AOM, DSS, Lipopolysaccharide (LPS), Tween80, methylcellulose, MTT, and Griess reagent were obtained from Sigma-Aldrich. Matrigel and collagen I were from Corning. Emodin was purchased from Chengdu Herbpurify Company (China). Purity was 95–99% as assessed by HPLC. For immunohistochemical (IHC) staining, the following primary antibodies were used: anti-F4/80 (MF4800, Caltag) and anti-IκBα (Cat No.710128, Invitrogen), anti-Ki67 (ab16667, Abcam), anti-CD11b (ab133357, Abcam), anti-CD3 (ab5690, Abcam), anti-CD206 (ab64693, Abcam), anti-COX-2 (Cat No.610204, BD Transduction Laboratories), anti-CD31 (Neomarkers, RB-10333-P), and anti-Vimentin (Cat No.5741, Cell Signaling Technology). For flow cytometry analyses the following directly-labelled antibodies were used: anti-Ly6C-FITC (clone HK1.4, Biolegend), anti-Gr1-Pacific Blue (clone RB6-8C5, Biolegend), anti-Ly6G-APC (clone 1A8, Bio Legend), anti-CD11b APC-Cy7 (clone N418, eBiosciences, ThermoFischer Scientific), anti-CD4-FITC (clone RM4-5, Biolegend), anti-CD8-PE (clone 53-6.7, eBioscience).
8
Western Blotting and Immunocytochemistry Protocol
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For western blotting, anti-collagen XVII (ab184996) and HRP-conjugated secondary antibody (ab205718) were purchased from Abcam, MA, USA. The anti-pAKT (9271), anti-AKT (9272), anti-pmTOR (5536), anti-mTOR (2983), anti-pp70S6K (9234), anti-p4EBP1 (2855), and anti-β-actin (4970) antibodies were purchased from Cell Signaling Technology, MA, USA. The anti-Ki67 (ab16667, Abcam) and HRP-conjugated secondary antibodies (K4002, Dako, Japan) were used for immunocytochemistry.
9
Antibody Profiling for MAPK Signaling
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Antibodies used in this study included: anti-phosphoERK1/2 (#4370), anti-phosphoMEK1/2 (#9154), and anti-MEK1/2 (#9124) (Cell Signaling Technology); anti-BRAF (SAB5300503), anti-CRAF (SAB5300393), anti-FLAG (F3165) and anti-β-actin (A2228) (Sigma); anti-HA (MAB6875, Novus Biologicals); anti-ERK2 (sc-154, Santa Cruz Biotechnology); anti-ERK1/2 (A0229, AB clonal); anti-Ki67 (ab16667, Abcam); and HRP-labeled secondary antibodies (Jackson Laboratories). Vemurafenib and LY3009120 were purchased from Medchemexpress; and D-luciferin from Gold Biotechnology. All other chemicals were obtained from Sigma.
10
AnxA5 and Cardiac Inflammation Post-Infarction
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To evaluate AnxA5 accumulation in the infarct area paraffin section of the mid-infarct region of the heart were stained using antibodies against AnxA5 (anti-human annexin V, 3357–100, Biovision, Milpitas, CA, USA), while counterstaining was performed using haematoxylin.
For analysis of the cardiac inflammatory response paraffin sections of the mid-infarct region of the heart were stained using antibodies against macrophages (anti-Mac3, 550292; BD Pharmingen, San Diego, CA, USA) and to quantify the number of proliferating macrophages antibodies against proliferation marker Ki67 were used (anti-Ki67, ab16667; Abcam, Cambridge, UK). The number of macrophages and proliferating macrophages was expressed as a number per mm2 in the septum (2 areas), border zones (2 areas), and infarcted myocardium (3 areas).
For analysis of the cardiac inflammatory response paraffin sections of the mid-infarct region of the heart were stained using antibodies against macrophages (anti-Mac3, 550292; BD Pharmingen, San Diego, CA, USA) and to quantify the number of proliferating macrophages antibodies against proliferation marker Ki67 were used (anti-Ki67, ab16667; Abcam, Cambridge, UK). The number of macrophages and proliferating macrophages was expressed as a number per mm2 in the septum (2 areas), border zones (2 areas), and infarcted myocardium (3 areas).
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