Nel701a001kt

Manufactured by PerkinElmer

Sourced in United States

The NEL701A001KT is a laboratory equipment product from PerkinElmer. It serves as a core function in various scientific applications. The detailed specifications and intended use of this product are not available in an unbiased and factual format within the constraints provided.

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Lab products found in correlation

Ab6326, by Abcam (2 mentions) Ab290, by Abcam (2 mentions) Prb 160p, by Fortrea (2 mentions) Troma 1, by Developmental Studies Hybridoma Bank (2 mentions) S1700, by Agilent Technologies (1 mentions) Hoechst, by Merck Group (1 mentions) Goat anti hrp, by Jackson ImmunoResearch (1 mentions) Rabbit anti 5 ht, by Merck Group (1 mentions) Prolong gold with dapi, by Thermo Fisher Scientific (1 mentions) Goat anti s100a10, by R&D Systems (1 mentions) Radiance 2100 confocal microscope, by Bio-Rad (1 mentions) A11120, by Thermo Fisher Scientific (1 mentions) Anti digoxigenin hrp, by Roche (1 mentions) Ta180004, by OriGene (1 mentions) Megascript kit, by Thermo Fisher Scientific (1 mentions) Ab41834, by Abcam (1 mentions) A20216, by Thermo Fisher Scientific (1 mentions)

10 protocols using nel701a001kt

Immunohistochemical Staining for Ki67

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Tumor tissues were fixed in 2% paraformaldehyde (PFA), subsequently dehydrated, embedded in paraffin and sectioned at 4 µm. After deparaffinization, slides were washed with AD before antigen retrieval at pH = 9 for 15 min in a steamer (Target retrieval solution, DAKO, S1700). Next, slides were washed with PBS and 3% H₂O₂ in PBS for 10 min. Before 45 min blocking in TNB buffer [0.1 M Tris-HCl pH = 7.5; 0.15 M NaCl; 0.5% blocking reagent (FP1012, TSA-kit, Perkin Elmer, NEL701A001KT)], slides were washed with AD, PBS and Tris-buffered saline (TBS) (2 x 5 min), followed by overnight incubation with Ki67 primary antibody at 4°C in TNB (Table 2). The next day, slides were washed with TBS before incubation with the secondary antibody in TNB for 1 h at room temperature (Table 2). Next, samples were washed with TBS before 10 min incubation in fluorescein-tyramide (FITC) (TSA-kit, Perkin Elmer, NEL701A001KT) (1/50 in amplification diluent). After washing with TBS, tissue sections were stained with 1 µg/mL Hoechst for 10 min (Sigma), before washing in TBS and mounting with fluomount (Agilent). Images of the stainings were taken using the Zeiss Axio Imager.M2 microscope and analyzed with ImageJ software.

Vanhevel J., Verlinden L., Loopmans S., Doms S., Janssens I., Bevers S., Stegen S., Wildiers H, & Verstuyf A. (2022). The Combination of the CDK4/6 Inhibitor, Palbociclib, With the Vitamin D3 Analog, Inecalcitol, Has Potent In Vitro and In Vivo Anticancer Effects in Hormone-Sensitive Breast Cancer, But Has a More Limited Effect in Triple-Negative Breast Cancer. Frontiers in Endocrinology, 13, 886238.

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Multi-modal Developmental Gene Expression

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Digoxigenin-UTP-labeled antisense RNA probes were transcribed using MEGAscript Kit (Ambion) according to the manufacturer's instructions. Whole-mount in situ hybridizations were performed according to previously published methods (Ning et al., 2013 (link)). In particular, fluorescent in situ hybridization in immunostained embryos was conducted as previously described (Mao et al., 2021 (link)). Briefly, for the detection of cxcr4a and tie1 expression, Tg(nkx2.5:ZsYellow) embryos were first immunostained using affinity-purified anti-ZsYellow antibody (1:800; TA180004, Origene), and then subjected to in situ hybridization with digoxigenin-labeled cxcr4a or tie1 probe. Anti-digoxigenin-HRP (1:400; Roche, 11633716001) was used as primary antibody to detect the probes and the hybridization signals were visualized by incubating embryos with fluorescein tyramide (1:50; PerkinElmer, NEL701A001KT). For the detection of cxcl12b expression, Tg(sox17:GFP) embryos were fluorescently stained with anti-GFP (1:1000; A11120, Invitrogen) antibody. The resulting hybridization signals were similarly generated with cyanine 3 tyramide (1:50; PerkinElmer, NEL701A001KT).

Liu J., Zhang M., Dong H., Liu J., Mao A., Ning G., Cao Y., Zhang Y, & Wang Q. (2022). Chemokine signaling synchronizes angioblast proliferation and differentiation during pharyngeal arch artery vasculogenesis. Development (Cambridge, England), 149(23), dev200754.

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Multimarker Immunofluorescence Staining

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Sections from the control group were permeabilized with PBS containing 0.1% Triton X-100 (PBST) and 2% fetal bovine serum and incubated overnight at room temperature with primary antibodies to netrin-G1 (1:500 goat anti-Netrin-G1a, R&D Systems) or p11 (1:100 goat anti-S100A10, R&D Systems) and 5-HT (1:1000 rabbit anti-5HT, Sigma) or p11 and anti-phospho-histone H3 (PH3) (1:200 rabbit anti-phospho S10, Abcam). Slides were then washed in PBST before applying secondary antibodies. Incubation with secondary antibodies (1:800 anti-goat HRP and 1:800 anti-rabbit Rhodamine Red, Jackson ImmunoResearch Laboratories) occurred at room temperature for 2 h in the dark. Slides were again washed in PBST prior to amplification. For amplification, slides were incubated in Tyramide Signal Amplification-Fluorescein solution (1:50; PerkinElmer, NEL701A001 KT) for 10 min at room temperature in the dark. A final series of washes with PBST was performed and the slides were mounted using Prolong Gold with DAPI (to visualize nuclei; Life Technologies, P36931).

King J.R., Velasquez J.C., Torii M, & Bonnin A. (2017). Effect of Maternal ±Citalopram Exposure on P11 Expression and Neurogenesis in the Mouse Fetal Brain. ACS chemical neuroscience, 8(5), 1019-1025.

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Immunohistochemical and Western Blot Analysis of Ror2 Signaling

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Tissue and organoid sections were deparaffinized, rehydrated and subjected to Tris-EDTA antigen retrieval for 20 min in a microwave. Primary antibodies were applied overnight at 4 °C. Antibodies and concentrations were: Ror2 (1:100; Nt 2535-2835, Ror2-s; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), eGFP (1:250; ab290; Abcam, Cambridge, MA, USA), K8 (1:250; TROMA-1; Developmental Studies Hybridoma Bank), K5 (1:5,000; PRB-160P; Covance, Princeton, NJ, USA), BrdU (1:250; ab6326; Abcam), CC3 (1:200; 9661; Cell Signaling Technology (CST), Danvers, MA, USA) and pERM (1:500; CST). Tyramide amplification was performed for Ror2 detection according to the manufacturer's instructions (NEL701A001KT; PerkinElmer, Waltham, MA, USA). For western blotting, the following antibodies were used: Ror1 (1:1000, #4102; CST), Dvl2 (1:1000, #3216; CST), RhoA (1:1000, #2117; CST), Rac1/2/3 (1:1000, #2465; CST), CDC42 (1:1000, #2466; CST), c-jun (1:1000, #9265; CST), p-c-jun s63 (1:1000, #9261; CST).

Roarty K., Pfefferle A.D., Creighton C.J., Perou C.M, & Rosen J.M. (2017). Ror2-mediated alternative Wnt signaling regulates cell fate and adhesion during mammary tumor progression. Oncogene, 36(43), 5958-5968.

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Immunostaining of Cryosectioned Hearts

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Embryonic or neonatal hearts were fixed in 4% PFA for 1 hour at 4°C, washed in 1X PBS and dehydrated in 30% sucrose solution at 4°C O/N. The following day, hearts were embedded in OCT (Optical Cutting Temperature Compound, Fischer Health Care, Catalog: 4585), and stored at −80°C. 20pm cryosections were thawed at room temperature for 15 minutes followed by two consecutive washes in 0.5% PBT and incubated in blocking solution (5% Donkey serum, 0.5% PBT) for 1 hour. Primary antibodies were incubated at 4°C O/N. The following day, the slides were washed in 0.5% PBT, and incubated with corresponding fluorescent-dye-conjugated secondary antibody for 2–3 hours at room temperature followed by washes in 0.5% PBT. Cryosections were then mounted in Fluromount G prior to confocal imaging. For Cxcr4, staining was performed using Cy3 or fluorescein tyramide signal amplification (TSA, Perkin Elmer, Catalog: NEL701A001KT or NEL701A001KT).

D’Amato G., Phansalkar R., Naftaly J.A., Fan X., Amir Z.A., Coronado P.E., Cowley D.O., Quinn K.E., Sharma B., Caron K.M., Vigilante A, & Red-Horse K. (2022). Endocardium-to-coronary artery differentiation during heart development and regeneration involves sequential roles of Bmp2 and Cxcl12/Cxcr4. Developmental cell, 57(22), 2517-2532.e6.

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Immunofluorescent Staining of Vascular Markers

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Cells grown on glass coverslips or tissue sections were fixed and permeabilized. Endogenous peroxidase activity was quenched by treatment with 3% H₂O₂ for 15 min. Cells or tissue sections were blocked with normal serum for 30 min. The coverslips were then incubated with rabbit anti-CD31 (ab32457), mouse anti-vWF (ab194405), rabbit anti-Endomucin (ab230018), and rabbit anti-copGFP (PA5-22688) at 4 °C overnight. Horse raddish peroxidase (HRP)-conjugated secondary antibodies (DS-0001, PV-6001 and PV-9001 or PV-9003, Zhongshan Biotech) were applied at 37 °C for 1 h, and then immunoreactive cells were visualized with fluorescein amplification reagent (NEL701A001KT, Perkinelmer). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Confocal images were collected by a Radiance2100 confocal microscope (Bio-Rad).

Chang Z., Zhang Y., Liu J., Zheng Y., Li H., Kong Y., Li P., Peng H., Shi Y., Cao B., Ran F., Chen Y., Song Y., Ye Q, & Ding L. (2020). Snail promotes the generation of vascular endothelium by breast cancer cells. Cell Death & Disease, 11(6), 457.

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Whole Mount RNAscope Tissue Imaging

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For detection of RNA transcripts in whole mount fins, the RNAscope Multiplex Fluorescent Reagent Kit v2 (ACD Bio-techne, 323100) was used with the following probes: bglap-C1 (ACD Bio-techne, 519671), cyp26b1 (ACD Bio-techne, 571281-C2), entpd5 (ACD Bio-techne, 820491-C4), runx2a (ACD Bio-techne, 409521-C2), and c5aR1 (ACD Bio-techne, 859561-C2). Fins were fixed overnight in 4% PFA at 4°C. The next day, fins were washed with PBT (1× PBS with 0.1% Tween) 3 × 5 min each. After incubation in RNAscope hydrogen peroxide for 10 min, fins were rinsed 3× with PBT, treated with RNAscope protease plus for 20 min, and rinsed 3× with PBT. All these steps were performed at RT. Probes and probe diluent were prewarmed to 40°C and cooled down to RT before use. The fins were incubated with probes overnight at 40°C. The next day, fins were washed 3× with 0.2× SSCT, 12 min each at RT. All the steps mentioned from here on were followed by such washing steps. Fins were post fixed with 4% PFA for 10 min at RT and incubated with AMP1, AMP2, and AMP3 for 30, 15, and 30 min, respectively, at 40°C. To develop the signal, fins were incubated with the corresponding RNAscope Multiplex FL v2 HRP for 15 min at 40°C. TSA-fluorophore (Perkin Elmer NEL701A001KT) was used in a 1:1500 dilution in TSA buffer (RNAscope kit). Fins were incubated for 15 min at 40°C.

Sehring I., Mohammadi H.F., Haffner-Luntzer M., Ignatius A., Huber-Lang M, & Weidinger G. (2022). Zebrafish fin regeneration involves generic and regeneration-specific osteoblast injury responses. eLife, 11, e77614.

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Immunohistochemical and Western Blot Analysis of Wnt Signaling

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Tissue and organoid sections were deparaffinized, rehydrated, and subjected to Tris-EDTA antigen retrieval for 20 min in a microwave. Primary antibodies were applied overnight at 4°C. Antibodies and concentrations were: Ror2 (1:100; Nt 2535-2835, Ror2-s; Developmental Studies Hybridoma Bank), eGFP (1:250; ab290, Abcam), K8 (1:250; TROMA-1; Developmental Studies Hybridoma Bank), K5 (1:5,000; PRB-160P; Covance), BrdU (1:250; ab6326; Abcam), CC3 (1:200; 9661; Cell Signaling Technology (CST)), and pERM (1:500; CST). Tyramide amplification was performed for Ror2 detection according to manufacturer instructions (NEL701A001KT; PerkinElmer). For western blotting, the following antibodies were used: Ror1 (1:1000, #4102, CST), Dvl2 (1:1000, #3216, CST), RhoA (1:1000, #2117, CST), Rac1/2/3 (1:1000, #2465, CST), CDC42 (1:1000, #2466, CST), c-jun (1:1000, #9265, CST), p-c-jun s63 (1:1000, #9261, CST).

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Immunohistochemical Analysis of S6-RP and ID3

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Standard immunohistology was performed on free-floating sections. Therefore, PFA fixed brains were infiltrated with 30% sucrose in PBS for 48 hours, cut into 40 mm sections, and stored in cryoprotectant (25% Glycerol, 25% Ethylene Glycol, 50% Phosphate Buffer pH 7.8) at À20 C until further use. Tissue sections were incubated with primary antibodies at the following dilutions: rabbit anti-S6-RP (1:50; Cell Signaling #2217S) or rabbit anti-ID3 (1:100; Abcam #Ab41834). Bound primary antibody was visualized using donkey anti-Rabbit antisera conjugated with Alexa 488 (1:500; Invitrogen #A-20216) for S6-RP or the tyramide signal amplification kit (PerkinElmer #NEL701A001KT) for ID3. Pictures were taken on a confocal SPE microscope. We counted individual ID3 positive cells and quantified the mean gray value for S6-RP in the SVZ using the software ImageJ.

Zywitza V., Misios A., Bunatyan L., Willnow T.E, & Rajewsky N. (2018). Single-Cell Transcriptomics Characterizes Cell Types in the Subventricular Zone and Uncovers Molecular Defects Impairing Adult Neurogenesis. Cell reports, 25(9).

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In situ RNA Detection in Whole Mount Fins

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For detection of RNA transcripts in whole mount fins, the RNAscope Multiplex Fluorescent Reagent Kit v2 (ACD Bio-techne, 323100) was used with the following probes: bglap-C1 (ACD Bio-techne, 519671) and cyp26b1-C2 (ACD Bio-techne, 571281). Fins were fixed overnight in 4% PFA at 4°C. The next day, fins were washed with PBT (1x PBS with 0.1% Tween) 3 x 5 min each. After incubation in RNAscope Hydrogen Peroxide for 10 min, fins were rinsed 3 x with PBT, treated with RNAscope Protease Plus for 20 min, and rinsed 3 x with PBT. All these steps were performed at RT. Probes and probe diluent were pre-warmed to 40°C and cooled down to RT before use. The fins were incubated with probes overnight at 40°C. The next day, fins were washed 3x with 0.2x SSCT, 12 min each at RT. All the steps mentioned from here on were followed by such washing steps. Fins were post fixed with 4% PFA for 10 min at RT and incubated with AMP1, AMP2 and AMP3 for 30, 15 and 30 min, respectively, at 40°C. To develop the signal, fins were incubated with the corresponding RNAscope Multiplex FL v2 HRP for 15 min at 40°C. TSA-fluorophore (Perkin Elmer NEL701A001KT) was used in a 1:1500 dilution in TSA buffer (RNAscope kit). Fins were incubated for 15 min at 40°C.

Sehring I.M., Haffner‐Luntzer M., Ignatius A., Huber‐Lang M., & Weidinger G. (2022). Zebrafish fin regeneration requires generic and regeneration-specific responses of osteoblasts to trauma.

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