For HE assay, the slices of tumor tissues were stained with hematoxylin for 5 min, then washed for 1 min, and restored to blue by 1% ammonia (30 s). Then, slices were swashed with running water (1 min). After, slices were stained by 0.5% HE or 1 min, flushed for 30 s then transparentized and finally mounted with neutral gum. For IHC assay, the appropriate antibody diluent and antigen repair treatment methods were selected following the manufacturer’s instructions. The paraffin-embedded tissue was dewaxed in xylene, dehydrated in ethanol, and 3% H2O2 blocked for 10 min. The antigen was extracted in 95°C EDTA buffer for 15–20 min. Sections were incubated with Ki67 antibody (Santa Cruz, USA) overnight at 4°C. After washing 3 times with PBS, sections were incubated with the secondary antibody coupled to HRP (Santa Cruz Inc., USA) at 37°C for 50 min. Finally, DAB chromogen (Beyotime Inc., China) was added, and then slices were washed with water and stained with hematoxylin.
Ki67 antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Ki67 antibody is a laboratory reagent used in biomedical research. It is a protein that binds to the Ki67 protein, which is a cellular marker for proliferation. The Ki67 antibody can be used to detect and quantify the presence of Ki67 in biological samples, providing information about cell growth and division.
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Lab products found in correlation
Dab chromogen, by Beyotime (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Apotome microscope, by Zeiss (1 mentions)
Sca 1, by Abcam (1 mentions)
Notch1, by Abcam (1 mentions)
Gata4, by Santa Cruz Biotechnology (1 mentions)
C kit h 300, by Santa Cruz Biotechnology (1 mentions)
Total irak1 antibody, by Santa Cruz Biotechnology (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Mouse monoclonal pcna, by Santa Cruz Biotechnology (1 mentions)
Vectashield antifade mounting medium with dapi, by Vector Laboratories (1 mentions)
Eclipse te2000 u, by Nikon (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Infinite m1000, by Tecan (1 mentions)
Wnt3a, by Merck Group (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Tgf β1, by Merck Group (1 mentions)
Ly294002, by Merck Group (1 mentions)
Fh535, by Merck Group (1 mentions)
P akt1, by Abcam (1 mentions)
26 protocols using ki67 antibody
1
Histology and Immunohistochemistry Assays
2
Metformin Inhibits Tumor Growth
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Institutional Animal Care and Use Committee of National Institute of Immunology, New Delhi, India approved all the animal studies. The right flank of female nude mice (nu/nu) was subcutaneously injected with 4 × 106 cells/0.15 ml of DMEM. Post one week of injection, the mice were administered metformin (5 mg/ml) in the drinking water. The tumor size was evaluated for 30 days. Tumor volume was measured with a caliper and calculated using the formula: (widest diameter × smallest diameter2 (link))/2. Post 30 days of injection, the mice were euthanized using carbon dioxide, followed by tumor excision and its weight determination. Tumor extracts were then prepared and immunoblotting was performed as described earlier. For Ki67 staining, tumors were fixed in formalin, washed with PBS, and embedded in paraffin for sectioning. Five micrometer sections were dewaxed and immunostained with Ki-67 antibody (Santa Cruz). Secondary detection was performed using anti-mouse Alexa Fluor 555 (Molecular Probes). Counterstaining was done with DAPI. The slides were imaged using a Zeiss ApoTome microscope and images were analyzed with the Zen Blue software.
3
Phenotypic Characterization of CPCs
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CPCs were passaged 3–5 times, fixed with 2% paraformaldehyde, and stained for c‐kit (H‐300; Santa Cruz Biotechnology, Dallas TX), GATA‐4 (9,053; Santa Cruz Biotechnology), and Nkx‐2.5 (14,033; Santa Cruz Biotechnology), Notch1 (ab8925, Abcam, Cambridge, UK), Sca‐1 (ab51317, Abcam), CD31 (sc‐28,188, Santa Cruz Biotechnology), or CD34 (7,324; Santa Cruz Biotechnology). Pooled patient cell lines were used, and the data were analyzed using Flow Jo V10 software. Cells were also analyzed before after ES with Ki‐67 antibody (sc‐23,900, Santa Cruz Biotechnology) and Calcein AM and Ethidium homodimer‐1 for live/dead quantification in addition to microscopy.
4
IRAK1 and Ki67 Immunohistochemistry in HCC
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About 33 HCC samples were collected from Eastern Hepatobiliary Surgery Hospital (Shanghai, China) and the procedure of human sample collection was approved by the Ethical Committee of Eastern Hepatobiliary Surgery Hospital. Immunohistochemical staining was performed with total IRAK1 antibody (Santa Cruz, CA, USA) and Ki67 antibody (Santa Cruz) as reported previously [8 (link)].
5
Immunohistochemical staining of xenograft tumors
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Paraffin-embedden xenograft tumor samples were cut into 4 µm thin slices. After deparaffinization and dehydration, endogenous peroxidase was inactivated by 2.5% H2O2 diluted in methanol. Tissue sections were subject to heat-induced antigen retrieval in 0.01 mmol/L sodium citrate buffer for 30 min. Slides were blocked in 20% (v/v) normal horse serum in phosphate buffer solution (PBS) and subsequently incubated with Ki-67 antibody (Santa Cruz). The immuno-signals were exhibited by a 3,3′-diaminoberzidine (DAB) substrate kit (Dako). All sections were counterstained with haematoxylin to show nuclei.
6
Evaluating Pancreatic Cancer Cell Proliferation
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Following dissection, the tumor tissues were fixed immediately in 10% phosphate-buffered formalin and were embedded in paraffin. Proliferation of pancreatic cancer cells was estimated by IHC staining with either a mouse monoclonal PCNA or Ki-67 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and the number of PCNA- and Ki-67-positive cells in a population of 200 cells sampled at × 400 magnification was counted. The proliferating cell nuclear antigen labeling index and Ki-67 labeling index were subsequently calculated. After treatment with mogroside V, vascularization was evaluated in CD31- and VEGF-immunostained tissue sections. The MVD was calculated according to Weidner's method.33 (link), 34 (link) TUNEL immunohistochemistry was performed using an In Situ Cell Death Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions.
7
Immunostaining of Cardiac Cell Transplants
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Rats were sacrificed at day 42 after cell injections. Hearts were harvested en bloc fixed in 10% neutral buffered formalin for 4 hours, cryoprotected in 30% sucrose overnight, and embedded in OCT medium. Immunostaining was performed per protocol. In brief, 5‐μm‐thick tissue sections were prepared starting at the heart apex using a Cryotome FSE Cryostat (Thermo Fisher Scientific Life Sciences). Tissue sections were cleared of OCT medium by washing in 1× PBS and then blocked with 3% bovine serum albumin containing 0.02% sodium azide and 0.1% Triton X‐100. Tissue sections were incubated with Alexa Fluor‐conjugated primary antibodies overnight at 4°C. Conjugated antibodies used were human‐specific Ku86 conjugated to Alexa Fluor 647 (sc‐5280; Santa Cruz Biotechnology) and PECAM1 conjugated to Alexa Fluor 568 (sc‐376764; Santa Cruz Biotechnology). All experiments performed included sections from control hearts incubated with the above‐conjugated antibodies. All sections were mounted with Vectashield antifade mounting medium with DAPI (Vector Laboratories) before imaging with an Olympus IX81 FluoView FV1000 confocal microscope. Ki‐67 antibody (sc‐23900; Santa Cruz Biotechnology) was used along with AF‐488 secondary antibody to stain CPCs fixed in 4% paraformaldehyde (See Supporting Information Fig. S1 ).
8
In Vivo Tumor Growth Inhibition
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In vivo tumor growth was analyzed in nude Balb/c mice (male, 4-week-old) that were randomly separated into four groups (n=4). To establish an in vivo tumor model, SW579 cells were treated with control shRNA (sh-NC), CCDC26 shRNA (sh-CCDC26), miR-422a inhibitor, or co-treated with CCDC26 shRNA (sh-CCDC26) and miR-422a inhibitor, respectively. Then, about 1×107 cells were subcutaneously injected into the mice. Tumor growth were measured every 7 days. After 35 days of injection, mice were sacrificed, and tumors were scaled. Tumor volume (V) was observed by measuring the length and width with calipers and calculated as measured with the method × 0.5. Ki-67 expression in tumor tissues was tested by immunohistochemical staining with the Ki67 antibody (Santa Cruz Biotechnology, USA). EZH2, Sirt6, and GAPDH protein levels in the tumor tissues were analyzed by Western blot using primer antibodies against EZH2 (1:1000) (Amyjet, China), Sirt6 (1:1000) (Amyjet, China), and GAPDH (1:1000) (Amyjet, China), respectively. All animal handling procedures were approved by Peking University Cancer Hospital and Institute Animal Welfare Committee (No. 76HG5573) and complied with the authorized Animal Care and Method Procedure published by this hospital and the World Medical Association.
9
Immunofluorescence Staining of MHC and Ki-67
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For MHC immunofluorescence, differentiated cells were fixed in 4% PFA buffered solution for 10 min and then blocked in 10% goat serum in PBS for 1 h. Cells were incubated overnight with sarcomeric MHC antibody (clone MF 20, Developmental Studies Hybridoma Bank), diluted 1:10 in 1% BSA PBS. Fluorescent conjugated secondary anti-mouse IgG1 antibody (Alexa488, Invitrogen) diluted 1:500 in 1% BSA PBS was used to detect the primary antibody. For Ki-67 immunofluorescence, growing cells at 24 h were fixed in 4% PFA buffered solution for 10 min, permeabilized in 0.2% Triton in PBS for 30 min, then blocked in 3% BSA in PBS for 20 min. Cells were incubated overnight with 1:100 Ki-67 antibody (Santa Cruz Biotechnology) in 0.5% BSA in PBS. Fluorescent conjugated secondary anti-goat antibody (Alexa 555, Invitrogen) diluted 1:200 in 0.5% BSA in PBS was used to detect the primary antibody. Nuclei were counterstained with [0.5 μg/ml] Hoechst and samples were mounted with 60% glycerol in Tris HCl 0.2M pH 9.3.
10
Xenograft Tumor Tissue Analysis
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The xenograft tumor tissue was fixed in 10% neutral paraformaldehyde for
24 hours. The tissues were then embedded in paraffin wax, sliced (5μm)
and stained with IHC. The Ki67antibody (Santa Cruz, California, USA)
was used to observe tumor growth. And the photographs were recorded
under a light microscope (Olympus, Tokyo, Japan, ×400).
24 hours. The tissues were then embedded in paraffin wax, sliced (5μm)
and stained with IHC. The Ki67antibody (Santa Cruz, California, USA)
was used to observe tumor growth. And the photographs were recorded
under a light microscope (Olympus, Tokyo, Japan, ×400).
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