A21271

Manufactured by Merck Group

Sourced in United States

The A21271 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use. The core function of this product is to provide a controlled and standardized environment for various scientific experiments and analyses. Further details about the intended use or specific applications of this product are not available.

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Lab products found in correlation

9 protocols using a21271

Immunohistochemistry of Embryonic Tissue Sections

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For immunohistochemistry embryos were fixed at the indicated stages in 4% PFA in 1 x PTW over night at 4 °C. Fixed embryos were either heated (Inoue and Wittbrodt, 2011 (link)) and mounted for cryo-sectioning or stained whole mount according to the protocols adapted from the zebrafish book (Westerfield, 2000 ). In case of sectioning, frontal or lateral cryosections (as indicated) of 16 μm thickness were prepared and stained as described in (Inoue and Wittbrodt, 2011 (link)). The following primary antibodies were used: anti-GS (mouse, Chemicon MAB302, 1:500), anti-Rx2 (rabbit, self-made, 1:250, anti-olRx2 antibody was raised against the full-length olRx2 (NP_001098373.1) recombinant protein in rabbits (Charles River), and affinity purified using the antigen coupled to AffiGel (Biorad) (Herder et al., 2013 (link)), anti-GFP (chicken, life technologies A10262, 1:500) and for anti-HuCD (mouse, invitrogen A-21271, 1:250) and anti-acetylated Tubulin (mouse, SIGMA T7451-100UL, 1:100). The following secondary antibodies were used at a 1:500 dilution, anti-mouse Cy5 (Jackson 715-175-151); anti mouse 546 (invitrogen A-11030), anti chicken 488 (Jackson 703-545-155), anti rabbit 549 (Jackson 112-505-144). DAPI (SIGMA, D956) nuclear counterstaining was performed (Inoue and Wittbrodt, 2011 (link)).

Sinn R., Peravali R., Heermann S, & Wittbrodt J. (2014). Differential responsiveness of distinct retinal domains to Atoh7. Mechanisms of Development, 133, 218-229.

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Immunocytochemical Analysis of Neural Markers

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Cells were fixed in PFA 4% for 30 min at room temperature and immunocytochemical procedures were carried out as previously described (Cortés-Campos et al., 2011 (link)). The following primary antibodies were used: rabbit anti-sGnRH BB8 (1:1000; Kah et al., 1986 (link)), rabbit anti-GFAP (1:200; Dako, Campintene, CA, USA; Z0334), chicken anti-vimentin (1:200; Millipore; AB5733), mouse anti-HUC (1:100; Invitrogen; A-21271), mouse anti-neurofilament (1:200; Sigma-Aldrich; N2787), mouse anti-sox2 (1:1000; Millipore; AB5603), mouse anti-nestin (1:100; BD Biosciences, San Jose, CA, USA; 611659) and mouse anti-PCNA (1:100; Sigma-Aldrich; P8825). The samples were DNA counter-stained with DAPI (1:1000; Invitrogen) and the reactivity revealed using Alexa-labeled secondary antibodies (1:500; Invitrogen).

Cortés-Campos C., Letelier J., Ceriani R, & Whitlock K.E. (2015). Zebrafish adult-derived hypothalamic neurospheres generate gonadotropin-releasing hormone (GnRH) neurons. Biology Open, 4(9), 1077-1086.

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Immunohistochemical Analysis of Mouse Brain

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Shortly after the behavioral or electrophysiology experiments, mice were sacrificed and perfused transcardially with phosphate buffered saline (PBS), followed by 4% paraformaldehyde (PFA) in PBS. Brains were retrieved, fixed in 4% PFA for 24 hr before being placed in a 30% sucrose solution. Brains were sliced at 30 μm thickness and prepared for the appropriate incubations, as described previously^{50 (link)}. Primary antibody included rabbit anti-GFP (1:1000, Millipore 06-896), Mouse anti-HuC/D (1:200, Molecular Probes A21271), mouse anti-NeuN (1:100, Millipore MAB377), rabbit anti-TH (1:1000, Pel-Freez P40101-0), at room temperature for 24 hours or at 4°C for 48 h when using rabbit anti-PV (1:1000, Swant PV 27). Secondary antibodies included Alexa Fluor 488-conjugated goat anti-chicken (1:500, Life Technologies), Alexa Fluor 488-conjugated goat anti-rabbit (1:500, Life Technologies), Alexa Fluor 568-conjugated donkey anti- rabbit (1:500, Life Technologies), Alexa Fluor 568-conjugated donkey anti-mouse (1:500, Life Technologies), Alexa Fluor 647-conjugated donkey anti-rabbit (1:500, Life Technologies), or Alexa Fluor 647-conjugated donkey anti-mouse (1:500, Life Technologies).

Mastro K.J., Zitelli K.T., Willard A.M., Leblanc K.H., Kravitz A.V, & Gittis A.H. (2017). Cell-Specific Pallidal Intervention Induces Long-Lasting Motor Recovery in Dopamine Depleted Mice. Nature neuroscience, 20(6), 815-823.

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Assessing Cell Proliferation and Apoptosis in Zebrafish

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For EdU pulse labeling, Edu from the Click-iT Alexa Fluor 647 Imaging Kit (Life Technologies #C10340) was diluted to 2.5 mg/ml for retro-orbital injection. Each euthanized fish examined at 5 wpf was injected with 1 µl EdU solution, while 1.5 µl was used for 6 wpf fish. Fish were fixed 2 hours postinjection for cryo-sectioning; EdU detection was performed according to the manufacturer’s protocol.
Cryo-sectioning and immunofluorescence staining were performed as previously described.^{44 (link)} Antibodies against EGFP (Life Technologies #A6455 and #A11120), fibrillarin (Abcam #ab4566) and activated caspase-3 (BD Biosciences #559565) were used as primary antibodies. Secondary antibodies were conjugated with Alexa 488, 568, 647 (Life Technologies). DAPI (Life Technologies #S36973) was used for nuclear staining. Fluorescent images were taken by a Leica SP5X scanning confocal microscope at the Confocal and Light Microscopy core facility at Dana-Farber Cancer Institute.
Paraffin sectioning, H&E staining and immunohistochemistry with primary antibodies against TH (Pel-Freez #P40101), HuC/D (Life Technologies #A-21271) and synaptophysin (Millipore #MAB5258) were performed at the DF/HCC Research Pathology Core, and FACS at DFCI Flow Cytometry Core according to standard protocols.

Tao T., Sondalle S.B., Shi H., Zhu S., Perez-Atayde A.R., Peng J., Baserga S.J, & Look A.T. (2017). The pre-rRNA processing factor DEF is rate limiting for the pathogenesis of MYCN-driven neuroblastoma. Oncogene, 36(27), 3852-3867.

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EdU Pulse Labeling and Tissue Analysis

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For EdU pulse labeling, EdU from the Click-iT Alexa Fluor 647 Imaging Kit (Life Technologies #C10340) was diluted to 2.5 mg/ml for retro-orbital injection. Each killed fish examined at 5 wpf was injected with 1 μl EdU solution, whereas 1.5 μl was used for 6 wpf fish. Fish were fixed 2 h post injection for cryo-sectioning; EdU detection was performed according to the manufacturer’s protocol.
Cryo-sectioning and immunofluorescence staining were performed as previously described.^{44 (link)} Antibodies against EGFP (Life Technologies #A6455 and #A11120), Fib (Abcam #ab4566) and activated caspase-3 (BD Biosciences #559565) were used as primary antibodies. Secondary antibodies were conjugated with Alexa 488, 568, 647 (Life Technologies). DAPI (Life Technologies #S36973) was used for nuclear staining. Fluorescent images were taken by a Leica SP5X scanning confocal microscope at the Confocal and Light Microscopy core facility at Dana-Farber Cancer Institute.
Paraffin sectioning, H&E staining and immunohistochemistry with primary antibodies against TH (Pel-Freez #P40101, Rogers, AR, USA), HuC/D (Life Technologies #A-21271) and synaptophysin (Millipore #MAB5258, Billerica, MA, USA) were performed at the DF/HCC Research Pathology Core, and fluorescence-activated cell sorting at DFCI Flow Cytometry Core according to standard protocols.

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Immunolabeling of Retinal Cell Types

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The following primary antibodies were used: anti-EGFP (chicken; Life Technologies, A10262; 1:500), rabbit anti-Rx2 (Reinhardt et al., 2015 (link); 1:500), anti-tagRFP (rabbit; Evrogen, AB233; 1:500), anti-Pax6 (rabbit; Hiss Diagnostics, PRB-278P; 1:200), PKCα (rabbit; Santa Cruz, sc-208; 1:200), anti-GS (mouse; Chemicon, MAB302; 1:500), anti-Sox2 (rabbit; Genetex, GTX101506; 1:500), anti-HuC/D (mouse; Thermo Fisher, A21271; 1:500), anti-recoverin (rabbit; Millipore, AB5585; 1:500), anti-Zpr-1 (mouse; Zebrafish International Resource Center; 1:500), anti-DsRed (rabbit; Clontech, 632496; 1:500), anti-BrdU (rat; AbD Serotec, BU1/75; 1:200). The following secondary antibodies were used: anti-mouse Cy5 (Jackson ImmunoResearch, 715-175-151), anti-chicken 488 (Jackson ImmunoResearch, 703-485-155), anti-rat DyLight549 (Jackson ImmunoResearch, 112-505-143), anti-rabbit DyLight549 (Jackson ImmunoResearch), anti-mouse Alexa546 (Life Technologies, A-11030) and anti-rat Alexa633 (Life Technologies, A21094). DAPI (Sigma-Aldrich, D9564) nuclear counterstaining was performed as described by Inoue and Wittbrodt (2011) (link).

Pérez Saturnino A., Lust K, & Wittbrodt J. (2018). Notch signalling patterns retinal composition by regulating atoh7 during post-embryonic growth. Development (Cambridge, England), 145(21), dev169698.

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Whole-Mount Immunostaining of Embryonic Brains

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For whole-mount immunostaining, embryos were fixed in 4% paraformaldehyde with 1× PBS (phosphate-buffered saline) and 4% sucrose at 4°C overnight. The fixed embryos were washed with PBSTr (PBS + 0.5% Triton X-100), dissected and incubated in acetone at −20 °C for 7 min. Dissected brains were washed once with PBSTr and twice with PDT (PBS, 1% BSA, 1% DMSO, 0.5% Triton X-100), and incubated in 5% goat serum in PDT at RT for 2 h. The samples were incubated with the primary antibody solution at 4 °C overnight. After four washes with PBST, the tissues were incubated with secondary antibodies (1/250 dilution, Alexa Fluor 488 and/or Alexa Fluor 594 goat anti-mouse and/or goat anti-rabbit IgG (H + L), (Molecular Probes, Invitrogen). Following staining, tissue was cleared step-wise in a glycerol series and mounted for confocal imaging. The following antibodies were used: chicken anti-GFP (1:500, Abcam, ab13970); rabbit anti-Kaede (1:500, MBL Co. Ltd., PM012M); mouse anti-HuC/D (1:500, Invitrogen, A-21271); mouse anti-TH (1:500, Millipore, MAB318); mouse anti-NeuroD1(1:500, gift from the Hibi Lab).

Kidwell C.U., Su C.Y., Hibi M, & Moens C.B. (2018). Multiple zebrafish atoh1 genes specify a diversity of neuronal types in the zebrafish cerebellum. Developmental biology, 438(1), 44-56.

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Immunofluorescence of Neuronal Populations

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Following Pomc FISH, brains with the Synaptophysin-tdTomato (Ai34D) allele were processed for immunofluorescence, with overnight incubation in the cocktail of a rabbit red fluorescent protein (RFP) antibody (1:400 dilution; Rockland Cat# 600-401-379, RRID:AB_2209751) that recognizes the tdTomato protein, and either a mouse monoclonal HuC/D antibody (2 μg/ml concentration; ThermoFisher Cat# A21271, RRID:AB_221448) or a Guinea pig NeuN antibody (1:2K dilution; Millipore Cat# ABN90, RRID:AB_11205592), to confirm neuronal identity. The primary antibodies were detected with Cy3-conjugated anti-rabbit and Alexa Fluor 647-conjugated anti-mouse or anti-Guinea pig IgGs (1:200 dilution each; Jackson Immunoresearch, West Grove, PA). Sections were coverslipped with SlowFade™ Diamond Antifade Mountant with DAPI (Thermo Fisher).

S.u.r.b.h.i., Wittmann G., Low M.J, & Lechan R.M. (2021). Adult-born proopiomelanocortin neurons derived from Rax-expressing precursors mitigate the metabolic effects of congenital hypothalamic proopiomelanocortin deficiency. Molecular Metabolism, 53, 101312.

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Immunohistochemical Analysis of Mouse Brain

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