Transfected U2OS cells were fixed with 4% formaldehyde in 6 well μ-slides (Ibidi) 1× PBS for 30 minutes at 25°C and subsequently washed with ~200 μl PBS three times. Cells were then permeabilized with 0.2% Triton X-100 in PBS for 10 minutes and washed with ~200 μl PBS three times. Cells were blocked with 1% BSA in PBS for 10 minutes, washed with PBS three times, and then incubated with primary antibody for 1 h at 25°C. Cells were washed with PBS three times, and then stained with 300nM DAPI for 10 minutes. The cells were again washed with PBS three times and incubated with FITC-conjugated goat anti-mouse IgG for 30 minutes. Cells were washed with 1× PBS three times and visualized in 0.5 % pphenylenediamine (Sigma) in 20 mM Tris, pH 8.8 with 90 % glycerol. Cells were quantified using ImageJ and color was applied using Adobe Photoshop CS3. PHH3 staining was done with an anti Ser-10 histone H3 antibody (sc-8656-R, Santa Cruz Biotechnology). Validation for sc-8656-R is available from Santa Cruz Biotechnology.
Sc 8656 r
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-8656-R is a laboratory equipment product manufactured by Santa Cruz Biotechnology. It is designed for use in scientific research and analysis. The core function of this product is to provide a specific application or capability for researchers, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
P phenylenediamine, by Merck Group (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Eclipse 90i, by Nikon (1 mentions)
Ab150073, by Abcam (1 mentions)
Random hexamer, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Mouse anti mf20, by Developmental Studies Hybridoma Bank (1 mentions)
Alexa fluor 488 or 568 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Sytox orange, by Thermo Fisher Scientific (1 mentions)
Technovit 7100 resin, by Kulzer (1 mentions)
Ab16667, by Abcam (1 mentions)
α tubulin sc 23948, by Santa Cruz Biotechnology (1 mentions)
Sc 23877, by Santa Cruz Biotechnology (1 mentions)
Sc 56299, by Santa Cruz Biotechnology (1 mentions)
Sc 198, by Santa Cruz Biotechnology (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Prb 149p, by Fortrea (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Dab chromogenic reagent kit, by Boster Bio (1 mentions)
15 protocols using sc 8656 r
1
Immunofluorescence Staining of Transfected U2OS Cells
2
Immunofluorescence Staining of Transfected U2OS Cells
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Transfected U2OS cells were fixed with 4% formaldehyde in 6 well μ-slides (Ibidi) 1× PBS for 30 minutes at 25°C and subsequently washed with ~200 μl PBS three times. Cells were then permeabilized with 0.2% Triton X-100 in PBS for 10 minutes and washed with ~200 μl PBS three times. Cells were blocked with 1% BSA in PBS for 10 minutes, washed with PBS three times, and then incubated with primary antibody for 1 h at 25°C. Cells were washed with PBS three times, and then stained with 300nM DAPI for 10 minutes. The cells were again washed with PBS three times and incubated with FITC-conjugated goat anti-mouse IgG for 30 minutes. Cells were washed with 1× PBS three times and visualized in 0.5 % pphenylenediamine (Sigma) in 20 mM Tris, pH 8.8 with 90 % glycerol. Cells were quantified using ImageJ and color was applied using Adobe Photoshop CS3. PHH3 staining was done with an anti Ser-10 histone H3 antibody (sc-8656-R, Santa Cruz Biotechnology). Validation for sc-8656-R is available from Santa Cruz Biotechnology.
3
Decellularized ECM Modulates Proliferation
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104 MC38 CRC cells were cultured for 48 h with 0.1 mg of decellularized 3D ECM fragments from WT tumors, Ccr2−/− tumors, or normal colon or without ECM fragments. Cells were subsequently fixed and stained according to a standard manufacturer protocol with a primary antibody against phosphohistone H3 (sc-8656-R; Santa Cruz Biotechnology, Inc.) and then with a fluorescently labeled secondary antibody (ab150073; Abcam) and DAPI. Cells were viewed under a fluorescent microscope (eclipse 90i; Nikon), and pictures were taken with a digital camera (1310; DVC). The fraction of phosphohistone H3+ cells out of total DAPI+ cells was calculated in randomly chosen fields using ImageJ software.
4
Validating c9hxorf36 Knockdown Efficiency
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A splice-blocking MO targeting the exon 2–intron 2 boundary of c9hxorf36 was designed and obtained from Gene Tools (5′-AAGCCACAAATTCAGACCTTCACCA-3′). The efficiency of the MO was confirmed by RT-PCR (reverse transcription–polymerase chain reaction). RNA was extracted from MO-injected embryos and controls at 3 dpf using TRIzol (Invitrogen). First-strand cDNA was synthesized using SuperScript III reverse transcriptase and random hexamers (Invitrogen), according to the manufacturer's instructions. We carried out PCR amplification using whole-embryo cDNA as template, and PCR products were subjected to direct DNA sequencing (Supplemental Fig. S2). One nanoliter of diluted MO (3, 6, and 9 ng) was injected into zebrafish embryos at the one- to four-cell stage. Injected embryos were fixed in 4% PFA (paraformaldehyde) overnight at 5.5 dpf (Alcian blue staining) and at 2 dpf for phospho-histone H3 staining. Alcian blue staining was performed to demarcate cartilage structures as described previously (Chassaing et al. 2012 (link)). Phospho-histone H3 immunostaining was performed as described previously (Golzio et al. 2012 (link)) using anti-histone H3 (ser10)-R, (sc-8656-R, Santa Cruz; 1:500 dilution). Embryos were imaged on a Nikon AZ100 microscope facilitated by NIS Elements software.
5
Immunofluorescent Staining of Zebrafish Embryos
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Zebrafish embryos were fixed 2 hours at room temperature in 4% paraformaldehyde, followed by dehydration and storage overnight in methanol at -20°C. Embryos were then digested in PBS containing 0.1% Tween20 and 10 μg/mL proteinase K and blocked in PBS containing 0.1% Tween20 and 2% sheep serum.
The primary antibodies used were mouse anti-MF20 (1:20; Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242) and rabbit anti-H3S10p (used at 1.33 μg/mL; sc-8656-R, Santa Cruz). The secondary antibodies were a peroxidase conjugated AffiniPure goat anti-mouse (1:500; 115-035-003, Jackson ImmunoResearch) and an Alexa fluor goat anti-rabbit 546 (1:5,000; A-11010, Life Technologies).
The primary antibodies used were mouse anti-MF20 (1:20; Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242) and rabbit anti-H3S10p (used at 1.33 μg/mL; sc-8656-R, Santa Cruz). The secondary antibodies were a peroxidase conjugated AffiniPure goat anti-mouse (1:500; 115-035-003, Jackson ImmunoResearch) and an Alexa fluor goat anti-rabbit 546 (1:5,000; A-11010, Life Technologies).
6
Multifaceted Analysis of Zebrafish Development
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Zebrafish embryos aged specific developmental stages were fixed in 4% paraformaldehyde/PBS for 12 h at 4°C. After extensive washing in PBST, embryos were transferred to 100% methanol at -20°C for 2h and subsequently subjected to rehydration with PBST. After blocking with 3% BSA/PBST at room temperature for 60 min, embryos were incubated at 4°C overnight with 1:200 diluted primary antibodies as follows: rabbit anti-pH3 (sc-8656-R, Santa Cruz), rabbit anti-human phosphorylated ribosomal Protein S6 (Ser235/236) (GTX113542, GeneTex), rabbit anti-human CK8 (GTX110311, Genetex), rabbit anti-human CK13 (GTX109883, Genetex), rabbit anti-human MMP9 (GTX100626, GeneTex), and rabbit anti-human CENPF (GTX100212, Genetex). After extensive washing in PBST for 10 min, embryos were incubated with 1:500 diluted Alexa Fluor 488 or 568-conjugated secondary antibodies (Invitrogen) for fluorescent detection of immunoreactive signals. For histology, some stained embryos were further infiltrated and embedded in Technovit 7100 resin (Heraeus Kulzer). Samples were sectioned at 3 μm intervals and counter-stain with either Hoechst 33342 (Invitrogen) or SYTOX orange (Invitrogen) to visualize the nuclear position.
7
Immunofluorescence Staining of Keratinocytes
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Keratinocytes were grown on glass coverslips, fixed with 3,8% formaldehyde in PBS for 10 minutes and permeabilized with cold methanol for 5 minutes or fixed with cold methanol for 10 minutes. Paraffin-embedded skin sections were prepared following convential procedures. Samples were incubated for 1 hour (cells) or overnight (sections) in a wet chamber with antibodies against keratin 10 (sc-23877), phospho-histone H3 (sc-8656-R), cyclin A (sc-56299), cyclin E (sc-198), αTubulin (SC-23948) (all from Santa Cruz, CA, USA), involucrin (I-9018, Sigma-Aldrich, Inc), keratin 1 (PRB-149P, Covance, Vienna, VA, USA) or Ki67 (ab16667, Abcam, Cambridge, UK). Then, primary antibodies were revealed with Alexa Fluor-conjugated goat anti rabbit or anti mouse antibodies (Jackson ImmunoResearch, PA, USA). Samples were then incubated with 0.2 µg/ml DAPI (Vector Lab, Burlingame, CA). Coverslips were mounted with Prolong Gold Antifade Reagent (ThermoFisher Scientific, Waltham, MA) and cells were visualized and photographed with a Zeiss fluorescent microscopy. Immunofluorescence staining was scored by counting 400 cells.
8
Immunostaining of Phosphohistone H3
9
Whole-mount Immunohistochemistry of Planarian Nervous System
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Whole‐mount immunohistochemistry was performed as previously described (Cebria & Newmark, 2005 ). The following primary antibodies were used: anti‐VC‐1 (1:15,000; kindly provided by Professor K. Watanabe and H. Orii; Sakai et al, 2000 ); 3C11 (or anti‐SYNAPSIN; 1:25, Developmental Studies Hybridoma Bank; Cebria, 2008 ) and Sánchez Alvarado, 2002) and Anti‐Histone H3 phosphorylated at serine 10 (1:500; sc‐8656‐R from Santa Cruz).
10
Immunostaining and Cell Cycle Analysis of Cardiomyocytes
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