Every 5th free-floating section (200 μm apart) was treated with 2 M HCl for 30 min and then with 3% hydrogen peroxide for 10 min at room temperature. The sections were incubated with PBS containing 0.3% Triton X-100 and 3% normal goat serum for 45 min, followed by overnight incubation at 4°C with mouse monoclonal anti-BrdU antibody (1:1,000; Millipore, Billerica, MA, USA) or mouse monoclonal anti-DCX antibody (1:500; Santa Cruz, CA, USA). After several PBS rinses, the sections were incubated in biotin-labeled goat anti-mouse IgG antibody (1:500; Santa Cruz) under agitation. Immunoreactivities were visualized based on avidin-biotin horseradish peroxidase complex (ABC Elite; Vector Laboratories, Inc., Burlingame, CA, USA) using 3,3′-diaminobenzidine (DAB; Vector Laboratories). For Ki67 immunostaining, the sections were incubated with rabbit monoclonal anti-Ki67 antibody (1:500; Abcam, Cambridge, UK) followed by Cy3-conjugated goat anti-rabbit IgG antibody (1:200, Jackson ImmunoResearch Laboratories, PA, USA).
Anti ki67 rabbit monoclonal antibody
Manufactured by Abcam
Sourced in United Kingdom
Anti-Ki67 rabbit monoclonal antibody is a lab equipment product used to detect the Ki67 protein, a cellular marker for proliferation. This antibody is designed for use in immunohistochemistry applications.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (2 mentions)
Abc elite, by Vector Laboratories (1 mentions)
Cy3 conjugated goat anti rabbit igg antibody, by Jackson ImmunoResearch (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Alexa fluor 594 conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated anti guinea pig igg, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti glucagon antibody, by Abcam (1 mentions)
Paraformaldehyde solution, by Fujifilm (1 mentions)
Anti cd31 rabbit polyclonal antibody, by Abcam (1 mentions)
Immpact dab, by Vector Laboratories (1 mentions)
Alkaline phosphatase substrate kit 3, by Vector Laboratories (1 mentions)
Abc ap mouse igg kit, by Vector Laboratories (1 mentions)
Bx53 dp74 microscope, by Olympus (1 mentions)
Nb100 74396, by Novus Biologicals (1 mentions)
Alexa 488 or alexa 594 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Hard set mounting medium with dapi, by Vector Laboratories (1 mentions)
Polyclonal anti tle3 antibody, by Proteintech (1 mentions)
Abz 9000, by Keyence (1 mentions)
5 protocols using anti ki67 rabbit monoclonal antibody
1
BrdU, DCX, and Ki67 Immunostaining
2
Histological Analysis of Murine Pancreatic Islets
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Tissue samples from dissected mice at day 28 were fixed with 4% paraformaldehyde solution (Wako, Osaka, Japan) overnight and embedded in paraffin, and we used a microtome to prepare 5-μm-thick sections for hematoxylin–eosin (H&E) staining and an immunohistochemical analysis. For the immunohistochemical analysis, the sections were incubated with 10% goat serum for 30 min and then incubated with guinea pig polyclonal anti-insulin antibody, mouse monoclonal anti-glucagon antibody, rabbit polyclonal anti-CD31 antibody, or rabbit monoclonal anti-Ki67 antibody (all from Abcam, Cambridge, MA) overnight. The bound antibodies were detected by Alexa Fluor 488-conjugated anti-guinea pig IgG, Alexa Fluor 594-conjugated anti-mouse IgG, or Alexa Fluor 594-conjugated anti-rabbit IgG (Thermo Fisher Scientific) as secondary antibodies. The stained sections were mounted using ProLong gold antifade reagent with DAPI (Thermo Fisher Scientific). The Ki67-labeling index of the β cells in the neo-islet tissues was determined by calculating the ratio of Ki67-positive nuclei to insulin+-β cells in randomly selected fields of liver sections from 5 individual mice (n = 5).
3
Immunohistochemical Analysis of FOXP3 and CD8
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Immunohistochemical analyses of FOXP3 and CD8 expression were performed on 3-μm-thick formalin-fixed paraffin-embedded (FFPE) tissue sections using the following antibodies: anti-human FOXP3 mouse monoclonal (clone: 236A/E7; #ab20034; Abcam, Cambridge, UK), anti-human CD8 mouse monoclonal (clone: 4B11; NCL-L-cd8-4B11; Leica, Newcastle, UK), anti-human CXCR4 rabbit polyclonal (NB100-74396; Novus, Continental, CO), anti-human α-smooth muscle actine (SMA) mouse monoclonal (clone: 1A4; 412021; Nichirei Bioscience, Tokyo, Japan), anti-human PD-L1 rabbit monoclonal (clone: 28–8; #ab205921; Abcam), anti-Ki67 rabbit monoclonal antibody (clone: SP6; ab16667; Abcam). Elite ABC Rabbit kit and Elite ABC Mouse kit (PK-6101, PK-6102; Vector Laboratories, Burlingame, CA), and ABC-AP Mouse IgG kit (AK-5002; Vector Laboratories). Tissue sections were incubated in ImmPACT DAB (Vector Laboratories) and Alkaline Phosphatase Substrate kit III (Vector Laboratories) until the desired staining intensity developed. The sections were then counterstained with hematoxylin and mounted; FOXP3/CXCR4 double staining was conducted without counterstain. Positive and negative staining controls for all antibodies were carried out in parallel using tonsillar tissue. Stained sections were viewed under an Olympus BX53+DP74 microscope.
4
Immunocytochemical Analysis of B16 Cells
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B16 cells were incubated with primary antibodies at 4°C overnight following blocking/permeabilization with PBS containing 0.3% Triton X100 and 5% goat serum for 20 minutes at room temperature. The following antibodies were used for immunocytochemistry: polyclonal anti-TLE3 antibody (Proteintech), and CyclinD1 mouse monoclonal antibody (72-13G)(Santa Cruz, Santa Cruz, CA, USA). anti-Ki67 rabbit monoclonal antibody (ab92742, Abcam, Cambridge, UK). The target proteins were visualized using an Alexa 488- or Alexa 594-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA). ABZ-9000 (Keyence) microscope was used for these analyses. To visualize the cell nuclei, the cells were mounted with Hard Set Mounting Medium with DAPI (Vector laboratories, Burlingame, CA, USA) and to visualize the cellular skeleton, the cells were stained with Rhodamine Phalloidin (Thermo Fisher Scientific).
5
Evaluation of Spheroid Viability and Proliferation
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Cell viability was assessed using a live/dead viability kit (Thermo Fisher) based on the manufacturer's instruction. The live and dead cells were stained green and red, respectively. For histochemical analysis of the spheroids, thin paraffin sections of the spheroids at day 7 were prepared (thickness of 7 μm). For the HE (hematoxylin and eosin) staining, prepared sections were stained with hematoxylin and eosin. Immunohistochemical analysis of Ki-67, a nuclear protein used as a marker for proliferative cells, was also performed. After chemical fixation with 1% antigen retrieval buffer (100× citrate buffer, pH 6.0; Abcam, Cambridge, UK), the sample was blocked with 10% donkey serum in PBS for 30 min, followed by incubation with an anti-Ki-67 rabbit monoclonal antibody (Abcam) at 4 °C overnight. The sections were incubated with goat anti-rabbit IgG H&L (Alexa Fluor 594; Thermo Fisher) for 30-60 min. Finally, the sections were mounted using a ProLong gold antifade reagent with DAPI (Thermo Fisher). The ratios of the Ki-67-positive cells were evaluated from ∼150-300 cells in 5 randomly selected images (n = 4-5).
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