A21247

Manufactured by Thermo Fisher Scientific

Sourced in United States

The A21247 is a laboratory instrument designed for sample preparation and analysis. It features automated processing capabilities to enhance efficiency and reproducibility in various analytical workflows. The core function of this product is to enable consistent and reliable sample handling in a laboratory environment.

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Lab products found in correlation

36 protocols using a21247

Tracking Splenic B Cell Migration

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C57BL/6J mice were injected intravenously with splenic B cells purified by negative depletion from either Wnk1^fl/+ RCE or Wnk1^fl/− RCE radiation bone marrow chimeras and labeled with 1 µM CMFDA. After 1 h, spleens were harvested and embedded in optimum cutting temperature compound (BDH) and frozen. 8-µm sections were fixed with 4% PFA and blocked with 2% goat serum in PBS. To identify the white pulp, sections were stained with anti-MADCAM1 (1:50, MECA-367; eBioscience) and an Alexa Fluor 647–goat anti-rat IgG secondary antibody (1:400, A-21247; Life Technologies). Sections were also counter-stained with DAPI. Images were acquired using a Zeiss Axio Scan.Z1 slide scanner and analyzed by drawing regions of interest around the spleen and the white pulp areas and cells were counted using FIJI. Numbers of cells were first normalized to the red and white pulp area, respectively, and a ratio of cell density in the white pulp to red pulp was then calculated.

Hayward D.A., Vanes L., Wissmann S., Sivapatham S., Hartweger H., O’May J.B., de Boer L.L., Mitter R., Köchl R., Stein J.V, & Tybulewicz V.L. (2023). B cell–intrinsic requirement for WNK1 kinase in antibody responses in mice. The Journal of Experimental Medicine, 220(3), e20211827.

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Immunofluorescence Characterization of Liver

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IF was performed using standard techniques, with liver sections incubated overnight with the following primary antibodies: rabbit anti-GFAP 1∶1000 (Z0334, Dako), chicken anti-β-gal 1∶1000 (ab9361, Abcam), rabbit anti-SMA1∶200 (ab32575 Abcam), rabbit anti-CRBP1 1∶200 (sc30106, Santa Cruz), rabbit anti-VWF 1∶200 (A0082, Dako), Rat anti-CD-31 1∶100 (553370, BD Pharmingen), and Rat anti-F4/80 1∶200 (122603, Biolegend). Immune complexes were detected with the following secondary antibodies: Alexa 488 conjugated goat anti-Rabbit IgG (A11034, Life Technologies), Alexa 488 conjugated goat anti-rat IgG (A21208, Life Technologies), Alexa 647 conjugated donkey anti-rat IgG (A21247, Life Technologies), Alexa 594 conjugated donkey anti-chicken IgG (703-516-155, Jackson Immunoresearch), antibodies. Sections were mounted with VectaShield (H-1000, Vector Labs) and imaged with a Zeiss Axiovert 200 microscope. Confocal images generated using an Olympus FV1000 confocal microscope.

Bahrami A.J., Gunaje J.J., Hayes B.J., Riehle K.J., Kenerson H.L., Yeung R.S., Stempien-Otero A.S., Campbell J.S, & Mahoney WM J.r. (2014). Regulator of G-Protein Signaling-5 Is a Marker of Hepatic Stellate Cells and Expression Mediates Response to Liver Injury. PLoS ONE, 9(10), e108505.

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Brain Perfusion and Immunostaining Protocol

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Animals were perfused with 10 mL cold PBS followed by 15 mL 4% PFA. Brains were kept in PFA at 4 degrees for 2 hr, then washed 3 times for 15 min each in PBS while rotating. Samples cryopreserved for 24 hr in 30% sucrose in PBS at four degrees. 40 um sections were taken with a microtome. Each section washed with 0.5 mL goat blocking solution for 1 hr at four degrees, then overnight at four degrees with rat primary antibody for somatostatin in blocking solution (MAB354; Millipore; 1:100). The next day, sections washed 3 times for 15 min in PBS with 0.25% Triton X-100 (PBS-T) at room temperature while gently shaking. Sections washed with 0.5 mL in blocking solution containing goat anti-rat Alexa 647 secondary antibody for 1 hr (A21247; Life Technologies Corporation; 1:200). Sections washed 3 times for 15 min in PBS-T, then mounted on slides and coverslipped.

Naka A., Veit J., Shababo B., Chance R.K., Risso D., Stafford D., Snyder B., Egladyous A., Chu D., Sridharan S., Mossing D.P., Paninski L., Ngai J, & Adesnik H. (2019). Complementary networks of cortical somatostatin interneurons enforce layer specific control. eLife, 8, e43696.

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Immunohistochemistry Secondary Antibodies

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Secondary antibodies used for immunohistochemistry were goat anti-mouse (IgG 488, Invitrogen, A11029, 1:1,000), goat anti-rabbit (IgG 488, Invitrogen, A11034, 1:1,000), goat anti-rabbit (IgG 546, Invitrogen, A11035, 1:1,000), goat anti-mouse (IgG 546, Invitrogen, A11030, 1:,1000), goat anti-rat (IgG 647, Life Technologies, A21247, 1:1,000).

Moyon S., Frawley R., Marechal D., Huang D., Marshall-Phelps K.L., Kegel L., Bøstrand S.M., Sadowski B., Jiang Y.H., Lyons D.A., Möbius W, & Casaccia P. (2021). TET1-mediated DNA hydroxymethylation regulates adult remyelination in mice. Nature Communications, 12, 3359.

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Immunohistochemical Analysis of Tissue Samples

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FVMs were embedded in paraffin and cut into 3-μm sections. After removing the paraffin, the sections were rehydrated, blocked, and incubated with primary antibodies overnight. The next morning, the sections were incubated with the secondary antibodies for 30 min at room temperature. Nuclei were counterstained with Hoechst 33342 (H3570, 1:400 dilution; Life Technologies, Gaithersburg, MD). The primary antibodies were tenascin-C supplied by Juntendo University (20 μg/ml), α-smooth muscle actin (α-SMA, ab21027, 1:50 dilution; Abcam, Cambridge, MA), CD34 (NCL-L-END, 1:2000 dilution; Leica Biosystems, Newcastle, UK), glial fibrillary acidic protein (GFAP, Z0334, 1:500 dilution; Dako, Glostrup, Denmark), integrin α_V (AB1930, 1:1000 dilution; Millipore, Bedford, MA), smooth muscle myosin heavy chain SM1 (7599, 1:3000; YAMASA, Tokyo, Japan), and SM2 (7601, 1:400; YAMASA). The secondary antibodies were anti-mouse Alexa Fluor 488 (A11001, 1:800 dilution; Life Technologies), anti-rat Alexa Fluor 488 (A11006, 1:800 dilution; Life Technologies), anti-mouse Alexa Fluor 647 (A21247, 1:800 dilution; Life Technologies), anti-goat Alexa Fluor 647 (A21469, 1:800 dilution; Life Technologies), and anti-rabbit Alexa Fluor 647 (A21244: 1:200 dilution; Life Technologies). Sections were examined and photographed with a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan).

Kobayashi Y., Yoshida S., Zhou Y., Nakama T., Ishikawa K., Arima M., Nakao S., Sassa Y., Takeda A., Hisatomi T., Ikeda Y., Matsuda A., Sonoda K.H, & Ishibashi T. (2016). Tenascin-C promotes angiogenesis in fibrovascular membranes in eyes with proliferative diabetic retinopathy. Molecular Vision, 22, 436-445.

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Multimodal Immunofluorescence Imaging of LDs

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The following antibodies were used: mouse anti-IBA1 (1:100, Santa-Cruz Biotechnology, 1022-5), mouse anti-CD68 (1:100, eBioscience, 14-0688-22), rabbit anti-PLIN2 (1:30, LSBio, LS-C402989), rabbit anti-ADFP (1:500, Abcam, ab52356), rabbit anti-LC3 (1:500, Sigma-Aldrich, L7543; MBL #PM036), rat anti-LAMP1 (1:500, Abcam, ab25245), rat anti-MBP (1:250, Millipore, MAB386), rabbit anti-NF (1:100, Bio-Rad, Ab8135), rabbit anti-iNOS (1:100, Abcam, ab15323), and mouse anti-F4/80 (1:100, Bio-Rad, MCA497G). The used secondary Alexa Fluor antibodies were Alexa488
(1:500, Life Technologies, A21202), Alexa555 (1:500, Life Technologies, A21430), and Alexa647
(1:500, Life Technologies, A21247). BODIPY ® 493/503 (2µM, Thermo Fisher Scientific, D3922) was used to stain the LDs.

Loix M., Wouters E., Vanherle S., Dehairs J., McManaman J.L., Kemps H., Swinnen J.V., Haidar M., Bogie J.F.J, & Hendriks J.J.A. (2022). Perilipin-2 limits remyelination by preventing lipid droplet degradation. Cellular and molecular life sciences : CMLS, 79(10).

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Fixation and Immunostaining of Oocytes

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Oocytes were fixed for 30–60 min at 37 °C in 100 mM HEPES (pH 7; titrated with KOH), 50 mM EGTA (pH 7; titrated with KOH), 2% formaldehyde (methanol-free) and 0.2% Triton X-100, based on previously published methods51 (link). Fixed oocytes were incubated in PBS with 0.1% Triton X-100 overnight at 4 °C. Antibody incubations were performed in PBS, 3% BSA and 0.1% Triton X-100. Primary antibodies used were mouse anti-pericentrin (30, BD Biosciences 611815; 1:750), mouse anti-γ-tubulin (GTU88, Sigma T6557; 1:3,000), rat anti-tyrosinated-α-tubulin (YOL1/34, AbD Serotec MCA78G; 1:3,000) and mouse anti-PLK1 (35–206, Abcam Ab17056; 1:1,000). Secondary antibodies used were Alexa-Fluor-488-labelled anti-mouse (Molecular Probes A11029; 1:400) and Alexa-Fluor-647-labelled anti-rat (Molecular Probes A21247; 1:400). DNA was stained with 5 mg ml⁻¹ Hoechst 33342 (Molecular Probes H3570).

Clift D, & Schuh M. (2015). A three-step MTOC fragmentation mechanism facilitates bipolar spindle assembly in mouse oocytes. Nature Communications, 6, 7217.

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Immunofluorescence Staining of Oocytes

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Oocytes were fixed for 30-60 min at 37°C in 100 mM HEPES (pH 7; titrated with KOH), 50 mM EGTA (pH 7; titrated with KOH), 2% formaldehyde (methanol free) and 0.2% Triton X-100, based on previously published methods^{51 (link)}. Fixed oocytes were incubated in PBS with 0.1% Triton X-100 overnight at 4°C. Antibody incubations were performed in PBS, 3% BSA and 0.1% Triton X-100. Primary antibodies used were mouse anti-pericentrin (30, BD Biosciences 611815; 1:750), mouse anti-γ-tubulin (GTU88, Sigma T6557; 1:3000), rat anti-tyrosinated-α-tubulin (YOL1/34, AbD Serotec MCA78G; 1:3000) and mouse anti-PLK1 (35-206, Abcam Ab17056; 1:1000). Secondary antibodies used were Alexa-Fluor-488-labelled anti-mouse (Molecular Probes A11029; 1:400) and Alexa-Fluor-647-labelled anti-rat (Molecular Probes A21247; 1:400). DNA was stained with 5 mg ml⁻¹ Hoechst 33342 (Molecular Probes H3570).

Clift D, & Schuh M. (2015). A three-step MTOC fragmentation mechanism facilitates bipolar spindle assembly in mouse oocytes. Nature Communications, 6, 7217.

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Immunofluorescence Staining of Dissected Tissues

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Dissected tissues were fixed in 4% paraformaldehyde (PF) in 0.2% Triton X-100 PB (PBT) for 20 min following three washes in 0.2% Triton X-100 PBS (PBST) for 10 min each. Samples were blocked in 5% goat serum PBST for 20 min. Primary antibodies were incubated in PBST overnight at 4 °C. Titres for primary antibodies were as follows: mouse nc82 (1:50, DSHB), mouse anti-GFP (1:200, A11120, Invitrogen) and rabbit anti-GFP (1:500, A11122, Invitrogen), rat anti-CD8 (1:200, MCD0800, CALTAG), rabbit anti-NOMPA38 (link) (1:200) and rabbit anti-DsRed (1:200, 632496, Clontech). Secondary antibodies were incubated in PBST for 1 h at room temperature after three washes. Secondary antibodies were Alexa 488 anti-rabbit, Alexa 488 anti-mouse, Alexa 568 anti-mouse, Alexa 568 anti-rabbit and Alexa 647 anti-rat (1:400, A11034, A11029, A11031, A11011, A21247, Molecular Probes). Samples were mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA) and observed under a LSM 700 Zeiss confocal microscope (Jena, Germany).

Jeong Y.T., Oh S.M., Shim J., Seo J.T., Kwon J.Y, & Moon S.J. (2016). Mechanosensory neurons control sweet sensing in Drosophila. Nature Communications, 7, 12872.

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Immunostaining of HA-tagged Proteins in C. elegans

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Worms were washed from one 10 cm plate using S-buffer and snap-frozen in liquid nitrogen on top of a poly-D-lysine-coated (Sigma; #P7405) glass slide. Afterward, they were fixed for 20 min at −20 °C in a methanol:acetone 1:1 ratio solution. They were then rinsed twice in PBS + 0.1% Tween-20 and blocked for 1 h with a solution of 1% bovine serum albumin, 10% goat serum in PBS + 0.1% Tween-20. Slides were then incubated with 200-fold diluted rat anti-HA tag antibody (Roche; #10744700) in blocking solution for 2 h at RT. They were then washed three times, 10 min each, with PBS + 0.1% Tween-20 and incubated for 45 min in 500-fold diluted secondary antirat antibody in blocking solution (Invitrogen; #A21247). After three additional 10 min washes in PBS + 0.1% Tween-20, coverslips were mounted using mounting media ProLong Diamond Antifade Mountant (Invitrogen; #P36966) and incubated overnight at RT prior to imaging

Fischer F., Benner C., Goyala A., Grigolon G., Vitiello D., Wu J., Zarse K., Ewald C.Y, & Ristow M. (2022). Ingestion of single guide RNAs induces gene overexpression and extends lifespan in Caenorhabditis elegans via CRISPR activation. The Journal of Biological Chemistry, 298(7), 102085.

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