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6 protocols using ki67 clone tec 3

1

Immunohistochemical Analysis of Tissues

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Antibody stains were performed on paraffin sections. Tissues were fixed overnight in 4% paraformaldehyde (PFA), dehydrated by washing through an ethanol gradient series, washed in histoclear and embedded in wax. 4 µm sections were cut, treated with histoclear and rehydrated through an ethanol gradient series. Antigen retrieval was obtained by boiling the sections in citrate buffer (0.1 M sodium citrate pH6 and 0.05% Tween). Sections were incubated in PBS with 10% sheep serum and then incubated with primary and secondary antibodies in PBS with 1% sheep serum. Sections were counterstained with DAPI nuclear stain. The following antibodies were used for immunohistochemistry; SOX9 was a kind gift from Francis Poulat (CNRS, Montpellier, France, 1:2000), Ki67 (clone TEC-3, Dako, 1:20), E-Cadherin (BD Transduction Labs, 1:50). Secondary fluorescent antibodies were obtained from Molecular Probes and were used at a 1:500 dilution. Fluorescent images were visualized and collected on a Leica TCS-SP2 confocal microscope.
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2

Quantifying Myocardial Infarction Outcomes

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The infarcted area was determined in serial sections (5 μm) of the infarcted myocardium (10 sections per mouse) after staining with Gomori*s 1-step trichrome staining using Diskus software (Hilgers, Königswinter, Germany) and expressed as percentage of LV area. The number of neutrophils (naphthol-AS-D-chloroacetate esterase and anti-MPO, #RB-373-A; Thermo Scientific, Waltham, MA, USA) was determined 1 day after MI, whereas the endothelial cells (CD31, #M20; Santa Cruz, Santa Cruz, CA, USA) was determined in formalin-fixed serial sections (three per mouse, 200 μm apart) of the infarcted myocardium 4 weeks after MI. Positive-stained cells were numbered in six different fields from infarcted area per section and expressed as cells/mm2. Blood vessels positive for CD31 were quantified and expressed as CD31/mm2. Apoptotic (TUNEL, TMRred; Roche, Mannheim, Germany) and proliferating (Ki67, clone TEC-3; DAKO, Hamburg, Germany) cells were stained, counted and expressed as per cent from total cells (DAPI staining). Total Akt (Akt1/2 (N-19), sc-1619; Santa Cruz) and phosphorylated-Akt (phospho-Akt (Ser) (193H12), #4058S; Cell Signaling Technologies, Beverly, MA, USA) were stained using Dylight 488–conjugated secondary antibodies (Thermo Scientific).
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3

Immunohistochemical Analysis of Cell Proliferation and Protein Expression

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Paraffin-embedded tissue sections were stained immunohistochemically with antibodies against Ki67 (clone TEC-3, DAKO), p65-pho (Abcam) and AR-V7 (Precision). The primary antibody was incubated at the appropriate concentration (1:1000) for one hour at room temperature. The secondary antibody was incubated for 60 minutes. Slides were rinsed extensively in tap water, counterstained with Mayer’s hematoxylin and mounted. For quantitation of the cell proliferation, the cells were counted as positive for Ki67 when nuclear immunoreactivity was observed. Each tissue section was counted manually in three different areas to assess the Ki67 positive cells index. The data were then presented as number of Ki67 positive cells/400x microscope field. Expression of p65-pho and AR-V7 were measured by calculating the percentage of DAB-stained nuclear area over total nuclear area (labeling index) using ImmunoRate program.61
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4

Immunohistochemical Staining of Prostate Tissues

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Paraffin-embedded tissue sections were stained immunohistochemically with antibodies against GRP-R (Abcam), Ki67 (clone TEC-3, DAKO), AR (N-20, Santa Cruz), p65-pho (Abcam) and AR-V7 (Precision). The primary antibody was incubated at the appropriate concentration (GRP-R, 1:200; Ki67, 1:1000; AR, 1:1000; p65-pho, 1:1000; AR-V7, 1:200) for one hour at room temperature. The secondary antibody was incubated for 60 minutes. Slides were rinsed extensively in tap water, counterstained with Mayer's hematoxylin and mounted.
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5

Immunohistochemical Analysis of Cell Proliferation and Protein Expression

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Paraffin-embedded tissue sections were stained immunohistochemically with antibodies against Ki67 (clone TEC-3, DAKO), p65-pho (Abcam) and AR-V7 (Precision). The primary antibody was incubated at the appropriate concentration (1:1000) for one hour at room temperature. The secondary antibody was incubated for 60 minutes. Slides were rinsed extensively in tap water, counterstained with Mayer’s hematoxylin and mounted. For quantitation of the cell proliferation, the cells were counted as positive for Ki67 when nuclear immunoreactivity was observed. Each tissue section was counted manually in three different areas to assess the Ki67 positive cells index. The data were then presented as number of Ki67 positive cells/400x microscope field. Expression of p65-pho and AR-V7 were measured by calculating the percentage of DAB-stained nuclear area over total nuclear area (labeling index) using ImmunoRate program.61
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6

Immunostaining of Cell Fate Markers

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Cells were grown for on cover slips and either left untreated or treated with resveratrol or DMSO (0,01%). At the indicated time points cells were fixed in 4% PFA-PBS and used for immunostaining. First cells were permeabilized with 1% TritonX-PBS for 10 minutes and washed with PBS, subsequent incubation with primary antibodies Ki-67 (clone TEC-3, M-7249, Dako), H3K9me3 and Casp3p17 (07–523, Millipore) and γH2AX (05–636, Millipore) was performed overnight, at 4°C. Next day, cover slips containing cells were then washed and incubated for one hour at 37°C in the dark with the following secondary antibodies (AlexaFluor488 goat anti-rat, AlexaFluor488 goat anti-rabbit and AlexaFluor488 rabbit anti-mouse, (Invitrogen) diluted in 3% BSA in PBS/0.05% Tween. Slides were then washed and counterstained with DAPI for nuclear staining mounted and analysed with fluorescence microscope (Olympos) and photographs were taken by an attached digital camera system. The percentage of Ki-67 positive cells was calculated as: = (number of Ki67 positive cells) / (total number of cells in a field) X100.
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