Xenograft tumor samples were collected and fixed in 10% neutral-buffered formalin (ThermoFisher Scientific) overnight. Tumors were washed with PBS and then transferred to 70% ethanol followed by embedding, sectioning, and hematoxylin and eosin staining. For immunohistochemical staining, tissue sections were processed according to methods previously described (Gan et al., 2010 (link); Gan et al., 2006 (link)). The primary antibodies used for immunohistochemistry were anti-Ki-67 (D2H10) (1:500), Cell Signaling Technology, 9027 S) and anti-cleaved caspase-3 (1:500), Cell Signaling Technology, 9661 s). Images were obtained at 400× magnification on an Olympus BX43 microscope.
Anti ki 67 d2h10
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-Ki-67 (D2H10) is a monoclonal antibody that recognizes the Ki-67 protein, a cellular marker associated with proliferation. The Ki-67 protein is expressed during active phases of the cell cycle (G1, S, G2, and mitosis), but is absent in resting cells (G0). This antibody can be used in immunohistochemical applications to detect and quantify cell proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Neutral buffered formalin, by Thermo Fisher Scientific (2 mentions)
Bx43 microscope, by Olympus (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Immpress hrp detection kit, by Vector Laboratories (1 mentions)
Anti phospho histone h2ax, by Merck Group (1 mentions)
Anti 4 hne, by Abcam (1 mentions)
Ab46545, by Abcam (1 mentions)
Foetal bovine serum (fbs), by Sartorius (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Sc 2031, by Santa Cruz Biotechnology (1 mentions)
4 protocols using anti ki 67 d2h10
1
Xenograft Tumor Histopathological Analysis
2
RNAscope and Immunohistochemistry for IGF1, CXCL13, and Immune Markers
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RISH was done by manual RNAscope Chromogenic assays, using RNAscope 2.5 HD Assay—BROWN or Duplex Assay according to the manufacturer (ACDBio). Chromogenic probes were Hs-IGF1, Hs-CXCL13, Hs-C7, Hs-CXCL13-C2, Hs-IGF1R-C2, Hs-SRD5A2-C2, and Hs-AR-C2. Semiquantitative scoring of RNAscope assays (based on dots per cell, 0–4 scale) was done per ACDBio guidelines. Immunohistochemistry was done using Tris-EDTA antigen retrieval, with ImmPRESS HRP Detection Kit and ImmPRESS Duet Double Staining Polymer Kit (HRP/AP) (Vector Laboratories). Primary antibodies were anti–Ki-67 (D2H10, 1:400) (Cell Signaling Technology), anti-CD20 (L26, 1:400) (MilliporeSigma), and anti-CD3 (MRQ-39, 1:800) (MilliporeSigma).
3
Histopathological Analysis of Xenograft and PDX Tumors
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Xenograft tumor and PDX tumor samples were collected and fixed in 10% neutral-buffered formalin (ThermoFisher Scientific) overnight. Tumors were washed with PBS and then transferred to 70% ethanol followed by embedding, sectioning, and hematoxylin and eosin staining. For immunohistochemical staining, tissue sections were processed as previously described74 (link),75 (link). The primary antibodies used for immunohistochemistry were anti-4-HNE (1:400, Abcam, ab46545), anti-phospho-histone H2A.X (1:500, EMD Millipore, 05–636), anti-Ki-67 (D2H10) (1:500, Cell Signaling Technology, 9027 S). Images were obtained at 400× magnification on an Olympus BX43 microscope.
4
Cell Culture and Immunoblotting Assays
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HCT116 and RKO cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) or Eagle's Minimum Essential Medium supplemented with 10% foetal bovine serum (Biological Industries, Beit HaEmek, ISRAEL) at 37°C in a humidified atmosphere with 5% CO2. Cell lines were authenticated by examining their morphology and growth characteristics. Anti‐CDKN1A (A5952), anti‐β‐actin (AC038), anti‐TP53INP1 (A5952), anti‐p‐p53‐S46 (AP046), anti‐p‐p53‐S392 (AP0860), anti‐p‐p53‐S376 (AP0987), anti‐p‐p53‐S15 (AP0950), anti‐AMPKα (A12718), anti‐p‐AMPK (AP0116), and anti‐AK4 (19854) were purchased from ABclonal Technology Co. (Wuhan, Hubei, CN). Anti‐CHD7 (A301‐223A) were purchased from Bethyl Laboratories Inc. (Montgomery, AL, USA). Anti‐Ki67 (D2H10) was purchased from Cell Signaling Technology (Danvers, MA, USA). Anti‐FLAG (M2, F3165) and anti‐α‐tubulin (SAB4500087) were purchased from Merck KGaA (Darmstadt, Germany). Anti‐TP53 (SC‐126) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fluorescein (111‐095‐003 and 115‐095‐003) secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA) and horseradish peroxidase‐conjugated secondary antibodies (sc‐2030 and sc‐2031) were purchased from Santa Cruz Biotechnology. 4‐6‐Diamidino‐2‐phenylindole was purchased from Merck KGaA.
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