Dorsolateral skin from embryos were microdissected and placed on nucleopore filters (VWR, WHA 800281) and fixed overnight with 4%PFA at 4° C. Skin explants were blocked for 6 hours in 5% normal donkey serum, 1% bovine serum albumin and 0.5% Triton-X100 at RT. Explants were incubated overnight at 4° C in the following primary antibodies: chicken anti-RFP (1:250, NovusBio, NBP1–97371), rabbit anti-Sox2 (1:400, Abcam, ab97959), rabbit anti-Sox9 (1:500, Millipore, ab5535), goat anti-Pcadherin (1:500, R&D Systems, AF761), rabbit anti-RFP (1:500, Rockland, 600–401-379), Ki-67 Monoclonal Antibody (SolA15) (1:200, Invitrogen, 14–5698-82). Explants were then washed in 0.2% Tween20/PBS for 4–6 hours on a rotator and then incubated with the following secondary antibodies: Alexa Fluor 488-donkey anti-goat (1:200, Invitrogen, A11055), Alexa Fluor 568-donkey anti-rabbit (1:200, Invitrogen, A10042), Alexa Fluor 488-donkey anti-rabbit (1:200, Invitrogen, A21206), Alexa Fluor 647-goat anti-rat (1:200, Invitrogen, A-21247), Alexa Fluor 405-donkey anti-rat (1:200, Invitrogen, A-21208) overnight at RT. Washes were carried out for 6 hours with 0.2% Tween20/PBS. Hoechst (1:750 in PBS) was used for 45 min before mounting on slides with SlowFade Gold Antifade Mountant (Invitrogen, S36936).
Ki 67 monoclonal antibody sola15
Manufactured by Thermo Fisher Scientific
The Ki-67 Monoclonal Antibody (SolA15) is a laboratory reagent used to detect the Ki-67 protein, which is a marker of cellular proliferation. It is commonly used in immunohistochemistry and other applications to identify and quantify proliferating cells.
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Lab products found in correlation
Ab97959, by Merck Group (1 mentions)
Alexa fluor 568 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Slowfade gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Goat anti pcadherin, by R&D Systems (1 mentions)
Goat anti rat alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Wha 800281, by Avantor (1 mentions)
Rabbit anti rfp, by Rockland Immunochemicals (1 mentions)
Ab5535, by R&D Systems (1 mentions)
Alexa fluor 488 donkey anti goat, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Ab36823, by Abcam (1 mentions)
Ab2893, by Abcam (1 mentions)
Anti mouse cd31, by BD (1 mentions)
Af647 donkey anti rabbit, by Jackson ImmunoResearch (1 mentions)
Af488 donkey anti goat, by Jackson ImmunoResearch (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Carl Roth (1 mentions)
Apotome 2, by Zeiss (1 mentions)
4 protocols using ki 67 monoclonal antibody sola15
1
Immunostaining of Embryonic Skin Explants
2
Exosome Effects on Cell Proliferation and DNA Damage
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To evaluate the effects of exosomes on cell proliferation and DNA damage, HUVEC and H9c2 cells were seeded on 4-well chamber culture slides. After 72 hours of culture with non-irradiated-exo or irradiatedexo, the cells were washed with PBS and xed in 4% paraformaldehyde for 10 minutes. After blocking, the cells were incubated with KI67 Monoclonal Antibody (SolA15, Invitrogen), anti-53BP1 antibody (ab36823, Abcam), or anti-gamma H2A.X (ab2893, Abcam), followed by associated Alexa our 488-conjugated second antibody. Nuclei were stained with DAPI, and the positively stained cells were counted under uorescence microscopy with 200-fold magni cation, and 20 elds per section were randomly selected for quantitative counting.
3
Cryosectioning and Immunostaining of Mouse Heart
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Extracted upper parts of the hearts were fixed for 2 h in 4% paraformaldehyde (Carl Roth) at RT. These parts were then transferred to 30% sucrose solution in PBS and kept at 4 °C overnight to prevent ice crystal formation. Samples were then embedded in Tissue-Tek O.C.T. Compound (Sakura) and quickly frozen and stored at −80 °C until sectioning. In total, 4 μm cryosections were then washed in PBS and permeabilized in PBST-0.1% (PBS with 0.1% Triton X-100 (Sigma-Aldrich). Blocking was performed in 10% BSA in PBS, followed by 1 h incubation of primary antibody. Slides were washed three times for 5 min in PBS and incubated afterwards with secondary antibody for 30 min. Lastly, nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) staining (Roche, 1:10,000) and slides were mounted with ProLong Gold (Invitrogen, P10144). Used antibodies are listed here: Anti-Mouse CD31 (BD Biosciences, 1:100, 553370), Thrombospondin-4 Antibody (Novus Biologicals, 893655, 1:100) and Ki-67 Monoclonal Antibody (SolA15) (eBioscience, 14-5698-80, 1:100). AF488 donkey anti goat (Jackson Immuno Research, 1:200), and AF647 donkey anti-rabbit (Jackson Immuno Research, 1:200) and AF647 donkey anti-rat (Jackson Immuno Research, 1:200).
4
Immunofluorescence Staining for Ki-67
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Cells were washed with phosphate buffered saline and fixed in 10% buffered formalin for 30 min. Subsequently, the samples were incubated in 10% v/v horse serum for 1 h. Primary antibody incubation was performed at 4 °C overnight using a Ki-67 monoclonal antibody (SolA15), eBioscience™, (Cat. No. 14-5698-82, ThermoFisher Scientific). Biotinylated rabbit anti-rat antibodies (Cat. No. Ab6733, Abcam, MA, USA) were employed as secondary antibody and the samples were stained with Strep-FITC (Cat. No. S3762, Sigma-Aldrich). Cell nuclei were counterstained with DAPI (Cat. No. 32670, Sigma-Aldrich). The staining was evaluated under a fluorescence microscope ZEISS Apotome.2 (Carl Zeiss, Jena, Germany). The negative control was performed by omitting the primary antibody incubation step.
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