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Anti ki67 sp6

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Anti-Ki67 (SP6) is an immunohistochemistry (IHC) primary antibody that targets the Ki67 protein, which is associated with cellular proliferation. It is commonly used in research and clinical settings to assess cell proliferation rates.

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Lab products found in correlation

Anti ttf1 8g7g3 1, by Agilent Technologies (3 mentions) Phospho s6, by Cell Signaling Technology (2 mentions) Phospho 4e bp1 thr37 46, by Cell Signaling Technology (2 mentions) Cleaved caspase 3 asp175 5a1e, by Cell Signaling Technology (2 mentions) S1699 retrieval buffer, by Agilent Technologies (2 mentions) Mouse human fcr blocker, by Miltenyi Biotec (2 mentions) Dab staining kit, by Thermo Fisher Scientific (2 mentions) Anti mouse igg2b, by Thermo Fisher Scientific (1 mentions) Anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions) Eclipse ti2, by Nikon (1 mentions) Phospho ampkα thr172 40h9, by Cell Signaling Technology (1 mentions) Hexokinase 2, by Cell Signaling Technology (1 mentions) Cleaved caspase 3 asp175, by Cell Signaling Technology (1 mentions) Tissue studio, by Definiens (1 mentions) Ab10983, by Abcam (1 mentions) E4382, by Merck Group (1 mentions) Oil red o, by Merck Group (1 mentions) Formaldehyde, by Merck Group (1 mentions) Bx51 microscope, by Olympus (1 mentions) Control mouse igg sc 2025, by Santa Cruz Biotechnology (1 mentions)

12 protocols using anti ki67 sp6

1

Immunofluorescence Staining of Muscle Cells

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Cells were fixed using 4% paraformaldehyde (Alfa Aesar, 43368) for 10 min and incubated in blocking PBS solution containing 2% BSA (AppliChem, 9048-46-8) and 0.2% Triton X-100 (Sigma-Aldrich, 9002-93-1) for 30 min to permeabilize cell membrane and block unspecific antigen binding. As the next step, the cells were incubated with primary antibodies in blocking solution for 2 hours, followed by 30 min of incubation with secondary antibodies and 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining (1:1000; Thermo Fisher Scientific, 62248). Stained cells were imaged using a Nikon microscope (ECLIPSE Ti2). The following primary antibodies have been used in this study: anti-human/mouse/rat/chicken Pax7 (5 μg/ml; R&D Systems, MAB1675), anti-human/mouse Myod1 (5.8A) (1:100; Thermo Fisher Scientific, MA512902), anti-mouse MyHC (1:1000; R&D Systems, MAB4470), and anti–Ki-67 (SP6) (1:250; Thermo Fisher Scientific, MA514520). The following secondary antibodies were used in this study at 1:500 dilution: anti-mouse immunoglobulin G1 (IgG1; goat, Alexa Fluor 647) (Thermo Fisher Scientific, A21240), anti-mouse IgG2B (goat, Alexa Fluor 546) (Thermo Fisher Scientific, A21240), and anti-rabbit IgG (H + L) (donkey, Alexa Fluor 546) (Thermo Fisher Scientific, A10040).
Kim I., Ghosh A., Bundschuh N., Hinte L., Petrosyan E., von Meyenn F, & Bar-Nur O. (2022). Integrative molecular roadmap for direct conversion of fibroblasts into myocytes and myogenic progenitor cells. Science Advances, 8(14), eabj4928.
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2

Immunohistochemical Analysis of Lung Cancer

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Tissue isolation, fixation and staining procedures were performed as previously described in Shackelford et al., 2013. Briefly, the following antibodies were used: phospho-4E-BP1 (Thr37/46) (Cell Signaling Technology, #2855 1:1600), cleaved caspase-3 (Asp175) (Cell Signaling Technology, #9661 1:200), anti-p63 (4A4) (abcam, #ab111449 1:100), anti-TTF-1 (8G7G3/1) (Dako, 1:200), anti-Ki67 (SP6) (Thermo Scientific, #RM-9106-S0 1:200), phospho-S6 (Cell Signaling Technology, #4585 1:400), hexokinase II (Cell Signaling Technology, #2867 1:800), phospho-AMPKα (Thr172) (40H9) (Cell Signaling Technology, #2535 1:100), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP (Cell Signaling Technology, #4370 1:400), phospho-GSKα/β (Ser21/9) (Cell Signaling Technology, #9331 1:50). Immunostained slides were digitally scanned onto a ScanScope AT (Aperio Technologies, Inc., Vista, CA). A pathologist blindly evaluated the H&E stained sections for distribution of histological subtypes, verified by p63 and TTF-1 immunostaining, and calculated the percentage of positively stained cells for Ki67, cleaved caspase-3, phospho-S6, and hexokinase II. Digital slides were analyzed with the Definiens’ Tissue Studio (Definiens Inc. Parsippany, NJ) to determine adenocarcinoma tumor area and necrotic tumor area.
Momcilovic M., McMickle R., Abt E., Seki A., Simko S.A., Magyar C., Stout D.B., Fishbein M.C., Walser T.C., Dubinett S.M, & Shackelford D.B. (2015). Heightening energetic stress selectively targets LKB1-deficient non-small cell lung cancers. Cancer research, 75(22), 4910-4922.
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3

Histological Analysis of Liver and Adipose Tissue

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Formalin-fixed, paraffin-embedded liver or adipose tissue samples (4.5-μm-thick sections) were stained with hematoxylin (51275; Sigma-Aldrich) and eosin (E4382; Sigma-Aldrich) or Sirius red. Immunohistochemistry for Ki-67 (anti-Ki67 SP6; Thermo Scientific) or UCP-1 (anti-UCP-1) (ab10983; Abcam, Cambridge, United Kingdom) staining were performed as described in in Slijepcevic et al.36 (link) From the snap-frozen liver tissue, 5-μM-thick cryosections were fixed with 3.7% formaldehyde (Sigma-Aldrich) for 60 minutes and stained with Oil Red O (Sigma-Aldrich) for 30 minutes. Digital imaging of all sections was performed using an Olympus BX-51 microscope (Olympus, Tokyo, Japan). The amount of liver fat stained by Oil Red O was calculated from 4 random pictures taken from each cryosection using ImageJ version 1.50i software (National Institutes of Health, Bethesda, MD).
Donkers J.M., Roscam Abbing R.L., van Weeghel M., Levels J.H., Boelen A., Schinkel A.H., Oude Elferink R.P, & van de Graaf S.F. (2020). Inhibition of Hepatic Bile Acid Uptake by Myrcludex B Promotes Glucagon-Like Peptide-1 Release and Reduces Obesity. Cellular and Molecular Gastroenterology and Hepatology, 10(3), 451-466.
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4

Antibody Use in Protein Detection

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The antibodies used in this study were as follows: anti-Myc (c3956; western blotting (WB):1:1,000, immunofluorescent (IF) staining:1:100) and anti-Flag (M2, F1804; WB:1:1,000) were purchased from Sigma; anti-β-gal (559761; IF:1:100) was from MP Cappel; anti-Ki-67 (Sp6: IF:1:150) was from Thermo Fisher Scientific Inc.; anti-Myc (9E10; IF:1:100) and control mouse IgG (sc-2025; IF:1:100) were from Santa Cruz; anti-LYVE1 (103-PA50; IF:1:100) was from Reliatech; anti-GAPDH (MAB374; WB:1:5,000) was from Millipore Bioscience Research Reagents; and anti-F4/80 (MCA497GA; IF:1:100) was from Serotec. Anti-P-YAP (no. 4911; WB:1:1,000) was purchased from Cell Signaling Technology. Anti-YAP (8G5; WB: 1:500; IF:1:50) and anti-Stab2 (IF:1:100) antibodies were established in our laboratory44 (link)45 (link). Actin in liver sections was stained using Alexa Fluor 488 phalloidin (A12379; IF:1:300; Thermo Fisher Scientific).
Miyamura N., Hata S., Itoh T., Tanaka M., Nishio M., Itoh M., Ogawa Y., Terai S., Sakaida I., Suzuki A., Miyajima A, & Nishina H. (2017). YAP determines the cell fate of injured mouse hepatocytes in vivo. Nature Communications, 8, 16017.
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5

Immunohistochemistry of Apoptosis Markers

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Deparaffinized and rehydrated formalin fixed samples tumor samples were subjected to antigen retrieval using Dako S1699 Retrieval Buffer at pH 6.1 at 90oC for 30 mins. After a 5 min peroxidase blockade, slides were blocked for 10 min at RT with PBS containing 0.5% BSA, 5% goat serum, and 1X mouse/human FcR blocker (Miltenyi Biotec 130–092-575). Slides were incubated with primary anti-Bnip3 (EPR4034, Abcam) at 1:100 at RT for 30 min, rabbit mAb to cleaved Caspase-3 (Asp175) (5A1E) (Cell Signaling Technology) or anti-Ki67 (SP6) (Thermo-Scientific) at 1:100 overnight at 4oC. IHC was visualized by DAB staining kit (TL-015-HD by Invitrogen), counterstained with hematoxylin, and mounted in non-aqueous solution (Richard-Allen Scientific 4112).
Nollet E.A., Cardo-Vila M., Ganguly S.S., Tran J.D., Schulz V.V., Cress A., Corey E, & Miranti C.K. (2020). Androgen Receptor-Induced Integrin α6β1 and Bnip3 Promotes Survival and Resistance to PI3K Inhibitors in Castration-Resistant Prostate Cancer. Oncogene, 39(31), 5390-5404.
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6

Immunofluorescence Analysis of Differentiating GSCs

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Briefly, differentiating GSCs were grown on 10 × 10 mm coverslips. Cells were then fixed in 3.7% formaldehyde and permeabilized with 0.5% Triton X-100. Then, cells were incubated with rabbit anti-GFAP (DAKO, Glostrup, Denmark) and anti-Ki67 (SP6, Thermo Scientific, Waltham, MA) or mouse anti-phospho histone H2AX (05–636, Millipore, Billerica, MA) antibodies. Texas red-conjugated or FITC-conjugated goat anti–rabbit or anti-mouse secondary antibodies (Jackson ImmunoResearch, Cambridgeshire, UK) were used for detection. For fluorescence quantification, markers were analyzed by using the AxioVision software (Carl Zeiss Inc.). Nuclear area was quantified using ImageJ software (NIH, Bethesda) on at least 100 DAPI-positive nuclei.
Ferrandez E., Gutierrez O., Segundo D.S, & Fernandez-Luna J.L. (2018). NFκB activation in differentiating glioblastoma stem-like cells is promoted by hyaluronic acid signaling through TLR4. Scientific Reports, 8, 6341.
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7

Immunohistochemical Analysis of Lung Samples

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After the fixation step in 10% neutral buffered formalin overnight, lungs were transferred to 70% ethanol and further processing and embedding was done by the TPCL at UCLA. The following antibodies were used: anti-CK5 (EP1601Y) (abcam, ab52635 1:100), anti-TTF1 (8G7G3/1) (Dako, 1:1000), anti-Ki67 (SP6) (Thermo-Scientific, RM-9106-S1 1:200), anti-Glut1 (alpha-diagnostic, GT11-A, 1:400), anti-Cleaved Caspase 3 (CST, #9664, 1:1000). Slides were scanned onto a ScanScope AT (Aperio Technlogies Inc.). Digital slides were analyzed with Definiens and QuPath software.
Momcilovic M., Jones A., Bailey S.T., Waldmann C.M., Li R., Lee J.T., Abdelhady G., Gomez A., Holloway T., Schmid E., Stout D., Fishbein M.C., Stiles L., Dabir D.V., Dubinett S.M., Christofk H., Shirihai O., Koehler C.M., Sadeghi S, & Shackelford D.B. (2019). In vivo imaging of mitochondrial membrane potential in non-small cell lung cancer. Nature, 575(7782), 380-384.
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8

Immunohistochemistry of Apoptosis Markers

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Deparaffinized and rehydrated formalin fixed samples tumor samples were subjected to antigen retrieval using Dako S1699 Retrieval Buffer at pH 6.1 at 90oC for 30 mins. After a 5 min peroxidase blockade, slides were blocked for 10 min at RT with PBS containing 0.5% BSA, 5% goat serum, and 1X mouse/human FcR blocker (Miltenyi Biotec 130–092-575). Slides were incubated with primary anti-Bnip3 (EPR4034, Abcam) at 1:100 at RT for 30 min, rabbit mAb to cleaved Caspase-3 (Asp175) (5A1E) (Cell Signaling Technology) or anti-Ki67 (SP6) (Thermo-Scientific) at 1:100 overnight at 4oC. IHC was visualized by DAB staining kit (TL-015-HD by Invitrogen), counterstained with hematoxylin, and mounted in non-aqueous solution (Richard-Allen Scientific 4112).
Nollet E.A., Cardo-Vila M., Ganguly S.S., Tran J.D., Schulz V.V., Cress A., Corey E, & Miranti C.K. (2020). Androgen Receptor-Induced Integrin α6β1 and Bnip3 Promotes Survival and Resistance to PI3K Inhibitors in Castration-Resistant Prostate Cancer. Oncogene, 39(31), 5390-5404.
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9

Prostate Cancer Cell Line Characterization

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The AR expressing, via lentiviral infection of LV-AR, hTERT immortalized human prostate epithelial 957E/hTERT cells (PrEC-AR) and c-Myc transformed PrEC-AR cells (PrEC-AR-c-Myc) were from Dr. John T. Issacs (Johns Hopkins University). Hi-MYC cell lines were purchased from AACR and cultured in DMEM supplemented with 10% FBS. PrEC-AR and PrEC-AR-c-Myc cell lines were cultured in Keratinocyte Serum Free Media supplemented with supplemented with bovine pituitary extract and recombinant epidermal growth factor (ThermoFisher) and 1% penicillin/streptomycin in 5% CO2 environment at 37°C. For in vitro experiments DZNep, GSK126, and EPZ6438 were all purchased from Xcessbio. For in vivo experiments DZNep was purchased from Sigma, and Epizyme Pharmaceuticals generously supplied EPZ011989. Antibodies used were anti-P53 (D2H9O, Cell Signaling), anti-EZH2 (D2C9, Cell Signaling), anti-H3K27me3 (C36B11, Cell Signaling), and anti-Ki67 (Sp6, Thermo Fisher). Mycoplasma testing is conducted every 3 months using standard PCR protocol from Sigma: Venor GeM Mycoplasma Detection Kit. Cell lines are authenticated using standard STR profiling.
Burkhart D.L., Morel K.L., Wadosky K.M., Labbé D.P., Galbo P.M., Dalimov Z., Xu B., Loda M, & Ellis L. (2020). Evidence that EZH2 Deregulation is an Actionable Therapeutic Target for Prevention of Prostate Cancer. Cancer prevention research (Philadelphia, Pa.), 13(12), 979-988.
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10

Tissue Analysis of Lung Tumors

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Tissue isolation, fixation and staining procedures were performed as previously described (Momcilovic et al., 2017 (link); Momcilovic et al., 2015 (link); Shackelford et al., 2013 (link)). Briefly, lungs or tumors were fixed overnight in 10% buffered formalin, and then transferred to 70% ethanol. Tissues were processed and embedded by TPCL at UCLA. The following antibodies were used: phospho-4EBP1 (Thr37/46) (Cell Signaling Technology, #2855 1:800), anti-CK5 (EP1601Y) (abcam, ab52635 1:100), anti-TTF1 (8G7G3/1) (Dako, 1:1000), anti-Ki67 (SP6) (Thermo Scientific, RM-9106-S0 1:200), phospho-S6 (Cell Signaling Technology, #4585 1:400), phospho-GSK3α/β (Ser21/9) (Cell Signaling Technology, #9331 1:50), c-Jun (Cell Signaling Technology, #9165 1:800), phospho-c-Jun (S73) (D47G9) (Cell Signaling Technology, #3270 1:200), anti-SLC1A5 (Sigma, HPA035240, 1:400), anti-GLUT1 (Alpha Diagnostic, GT11A, 1:400), anti-Myc (Y69) (abcam, ab32072, 1:100). Slides were scanned onto a ScanScope AT (Aperio Technologies, Inc., Vista, CA). Digital slides were analyzed with QuPath software in order to determine percent positive cells for Ki67, GLUT1 and p4EBP1 stains.
Momcilovic M., Bailey S.T., Lee J.T., Fishbein M.C., Braas D., Go J., Graeber T.G., Parlati F., Demo S., Li R., Walser T.C., Gricowski M., Shuman R., Ibarra J., Fridman D., Phelps M.E., Badran K., John M.S., Bernthal N.M., Federman N., Yanagawa J., Dubinett S.M., Sadeghi S., Christofk H.R, & Shackelford D.B. (2018). The GSK3 signaling axis regulates adaptive glutamine metabolism in lung squamous cell carcinoma. Cancer cell, 33(5), 905-921.e5.
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