A 21244

Manufactured by Thermo Fisher Scientific

Sourced in United States

The A-21244 is a laboratory instrument manufactured by Thermo Fisher Scientific. It is designed to perform precise and accurate measurements for a variety of applications. The core function of the A-21244 is to provide reliable data collection and analysis capabilities to support research and diagnostic processes.

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Lab products found in correlation

82 protocols using a 21244

Immunohistochemistry protocol for tongue and trigeminal ganglia

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Immunohistochemistry was performed as previously described.^{33 (link)} Tongues were flash frozen in OCT (Tissue-Tek) in liquid nitrogen. Trigeminal ganglia were fixed in 4% paraformaldehyde for 2 h, washed in PBS, and submerged in 30% sucrose. Sagittal tissue cryosections (25 μm) were prepared on slides. Sections were dried at 37°C for 1 h, and tongue sections were post-fixed in 4% paraformaldehyde for 15 min. Slides were washed in PBS and incubated in 5% normal goat serum (NGS) + PBST (PBS, 0.3% Triton X-100) at room temperature for 1 h. Sections were then incubated overnight in primary antibody mixed in NGS + PBST at 4°C. The next day, slides were washed three times in PBST and then incubated for 2 h in secondary antibody mixed in NGS + PBST. Following this, slides were washed five times in PBS, and then mounted in Fluoromount-G with DAPI (Southern Biotech).
Antibodies used in this study were chicken anti-GFP (1:1000 Abcam, ab13970, lot GR236651–25, RRID:AB_300798), rabbit anti-β3 tubulin (1:3000, Abcam, ab18207, lot GR3221401–3, RRID:AB_444319), rat anti-keratin8 (1:100, Developmental Studies Hybridoma
Bank, supernatant, RRID:AB_531826), Alexa 488 anti-chicken (1:1000, ThermoFisher, A-11039, RRID:AB_2534096), Alexa 594 anti-rat (1:1000, Fisher Scientific, A11007, RRID:AB_141374), Alexa 647 anti-rabbit (1:1000, Fisher Scientific, A21244, RRID:AB_2535812).

Moayedi Y., Xu S., Obayashi S.K., Hoffman B.U., Gerling G.J, & Lumpkin E.A. (2023). The cellular basis of mechanosensation in mammalian tongue. Cell reports, 42(2), 112087.

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Immunohistochemical Analysis of Liver Tissues

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Antibodies (Abcam) against desmin (ab15200; 1/200), c-RBP1 (ab154881; 1/500), α-SMA (ab124964, 1/1000) and Ki67 (ab16667; 1/500) were used for immunohistochemistry on 4 µm paraffin liver sections previously dewaxed and unmasked in sodium citrate buffer (0.1 M; pH 6). AlexaFluor647 conjugated secondary antibody (Fisher Scientific, A21244; 1/500) was then incubated for 1 h at RT and nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI; 0.3 µg/mL, Sigma-Aldrich, D1388). The slides were then examined under confocal fluorescence microscopy, (SP8 Leica Microsystems) at 400X magnification. Images were acquired using the software Leica Acquisition System X and treated using open source FiJi software.

Hoffmann C., Djerir N.E., Danckaert A., Fernandes J., Roux P., Charrueau C., Lachagès A.M., Charlotte F., Brocheriou I., Clément K., Aron-Wisnewsky J., Foufelle F., Ratziu V., Hainque B., Bonnefont-Rousselot D., Bigey P, & Escriou V. (2020). Hepatic stellate cell hypertrophy is associated with metabolic liver fibrosis. Scientific Reports, 10, 3850.

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Immunostaining Protocol for Mouse Brain

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At 0.5 or 3 months after injury, mice were transcardially perfused with 4% paraformaldehyde (PFA) and free-floating vibratome sections (50 µm) were processed using standard immunostaining procedures^{71 (link)}. Sections were stained with the following primary antibodies: GFP (1:1000; cat. no. GFP-1020, Aves Labs), NEUN (1:1000; cat. no. MAB377, Millipore), GFAP (1:500, cat. no. MAB3402, Millipore) and IBA1 (1:1000, cat. no. 019-19740, Fujifilm). Secondary antibodies were Alexa 488, 546, 594, and 647 (1:1000; cat. nos. A-11039, A-11005, A-11030 and A-21244, Fisher Scientific). Sections were then mounted on charged slides (Superfrost plus; Fisher Scientific) with Fluoromount-G containing DAPI (Southern Biotech). Images were obtained with a Leica DM6 epifluorescence microscope. Brightness and contrast were adjusted manually using Adobe Photoshop; z-stacks were generated using Leica software.

Frankowski J.C., Foik A.T., Tierno A., Machhor J.R., Lyon D.C, & Hunt R.F. (2021). Traumatic brain injury to primary visual cortex produces long-lasting circuit dysfunction. Communications Biology, 4, 1297.

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Immunofluorescence Staining of ALS-associated Proteins

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First, 5 × 10³ cells/chamber HAP1 cells were cultured in an 8-well slide and chamber (192-008, Watson, Tokyo, Japan). Cells were fixed with 4% PFA in PBS (26123-55, Nacalai Tesque), washed with PBS three times, and blocked with blocking buffer (5% skim milk with 0.3% triton X-100). Primary antibodies (anti-TLS/FUS (ab154141, Abcam, Waltham, MA, USA), anti-ZAP3 (A304-038A, Bethyl, Montgomery, TX, USA), anti-Matrin3 (A300-590A, Bethyl)) were diluted with blocking buffer at 1:1000, anti-TIA-1 (ab140595, Abcam) and anti-TDP-43 (12892-1-AP, Proteintech, Rosemont, IL, USA) were diluted at 1:250, and incubated at 4 °C for 16 h. Cells were washed for three times, and incubated with secondary antibodies (AlexaFlour 647 goat anti-rabbit IgG (A21244, Invitrogen) and Alexa Fluor 555 donkey anti-mouse IgG (A31570, Invitrogen), diluted with blocking buffer at 1:1000) at 37 °C for 1 h. Cells were washed again and mounted with Vectashield (H-1800, Vector Laboratories, Burlingame, CA, USA), and the cells were observed under fluorescent microscopy (BZ-X700).

Yoneda R., Ueda N, & Kurokawa R. (2021). m6A Modified Short RNA Fragments Inhibit Cytoplasmic TLS/FUS Aggregation Induced by Hyperosmotic Stress. International Journal of Molecular Sciences, 22(20), 11014.

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Immunofluorescent Staining of Eye Tissues

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Paraffin embedded sections of human eyes were deparaffinized and rehydrated with a graded alcohol series. Immunofluorescent staining was performed with antibodies (Abs) against human von Willebrandt factor (F3520, Sigma), human MMR (CD206, MAB2534, R&D Systems), human CD80 (ab53003 Abcam) or human ROCK1 (sc-17794, Santa Cruz Biotechnology) or ROCK2 (sc-1851, Santa Cruz Biotechnology) and identified with Alexa Fluor 488 (10μg/ml, A-11055; Invitrogen) or 647 (10μg/ml, A21244; Invitrogen) secondary Abs. On day 3 after laser injury, 10μm frozen sections of the posterior segment were prepared. The mouse eye sections were incubated with a rat anti-mouse F4/80 mAb (MCA497R, AbD Serotec) or CD11b (550282, BD Pharmingen), and subsequently with the secondary Ab. In monkey eyes, CD68 (goat pAb, sc-7082, Santa Cruz), vWF (rabbit pAb, A0082, DAKO), ROCK1 (mouse mAb, sc-17794, Santa Cruz) and ROCK2 (goat pAb, sc-1851, Santa Cruz) were stained. Images were obtained with a Leica microscope.

Zandi S., Nakao S., Chun K.H., Fiorina P., Sun D., Arita R., Zhao M., Kim E., Schueller O., Campbell S., Taher M., Melhorn M.I., Schering A., Gatti F., Tezza S., Xie F., Vergani A., Yoshida S., Ishikawa K., Yamaguchi M., Sasaki F., Schmidt-Ullrich R., Hata Y., Enaida H., Yuzawa M., Yokomizo T., Kim Y.B., Sweetnam P., Ishibashi T, & Hafezi-Moghadam A. (2015). ROCK-Isoform Specific Polarization of Macrophages Associated with Age-Related Macular Degeneration. Cell reports, 10(7), 1173-1186.

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Immunohistochemical Analysis of TGFBR3, EGLN3, and GPRC5A Proteins

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Sampling fixations was performed using 4% paraformaldehyde for 7 days and processed with decalcification by EDTA for 30 days. Fixed samples were embedded with paraffin and sectioned into 6‐mm thick slices. Staining was performed using anti‐TGFBR3 antibody (1:200, 2000‐1‐AP, Proteintech), anti‐EGLN3 antibody (1:500, 55,398–1, Sabbiotech) and anti‐GPRC5A antibody (1:200, 10,309‐1‐AP, Proteintech). Images were scanned by a high‐resolution digital slide scanner (VS‐200, Olympus). For immunofluorescence staining, sections were incubated with proteinase K (1:200, Solarbio) for 30 min, subsequently with Triton X‐100 (0.1%, Beyotime) for 30 min, and with 3% calf serum for 30 min. The primary antibodies, including anti‐PDGFRA (1:200, PB0822, BOSTER) and anti‐PROCR (1:1000, ab56689, Abcam), were used to incubate sections at 4°C overnight, and then the secondary antibodies (1:500, A11008, A21244, A21422, Invitrogen) were applied for 1 h at room temperature. Nuclei were stained with DAPI solution (0.1%, Beyotime). The immunostaining image was captured by the fluorescence microscopy system (CX43, Olympus) and analysed by the matching cellSens Dimension software (FCSnap) and Zen 2.3 (ZEISS).

Lin P., Yan P., Zhu J., Huang S., Wang Z., Hu O., Jin H., Li Y., Zhang L., Zhao J., Chen L., Liu B., He J., Gan Y, & Liu P. (2023). Spatially multicellular variability of intervertebral disc degeneration by comparative single‐cell analysis. Cell Proliferation, 56(10), e13464.

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Immunofluorescence of Centromere Proteins

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For immunofluorescence, cells were fixed in 95% methanol with 5 mM EGTA for 1 min. The following antibodies were used: mouse anti–α-tubulin DM1 (1:1,000; T6199; Sigma-Aldrich), human anti-centromere (CREST; 1:25; 15-234-0001; Antibodies, Inc.), rabbit anti–rat kangaroo-Mad2 (1:100; a gift from J. DeLuca and J. Mick), mouse and rabbit secondary antibodies conjugated to Alexa Fluor 488 and 647, respectively (1:500; A11001 and A21244; Invitrogen), and a human secondary antibody conjugated to DyLight 405 (1:100; 109-475-098; Jackson ImmunoResearch Laboratories, Inc.).

Kuhn J, & Dumont S. (2017). Spindle assembly checkpoint satisfaction occurs via end-on but not lateral attachments under tension. The Journal of Cell Biology, 216(6), 1533-1542.

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NMDA-R Expression on Bone Marrow-Derived Macrophages

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The abundance of the NMDA-R on the surfaces of BMDMs was determined by flow cytometry. Nonpermeabilized cells were labeled with NMDA-R GluN1 subunit-specific antibody (catalog PA3-102, Invitrogen, Thermo Fisher Scientific). Cell-associated PA3-102 was detected with Alexa Fluor 647–conjugated secondary antibody (catalog A21244, Invitrogen, Thermo Fisher Scientific). Control cells were treated with secondary antibody only. All data were analyzed using FlowJo Software version 10.7.1 (BD Biosciences).

Mantuano E., Zampieri C., Azmoon P., Gunner C.B., Heye K.R, & Gonias S.L. (2023). An LRP1-binding motif in cellular prion protein replicates cell-signaling activities of the full-length protein. JCI Insight, 8(15), e170121.

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Visualization of SIRT1 and PGC-1α in HepG2 Cells

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Cells were seeded and cultured on gelatin-coated glass coverslips. HepG2 cells were treated as indicated, incubated with 100nM MitoTracker Red (M7512, Invitrogen, CA), and fixed with 4% (w/v) paraformaldehyde in PBS at room temperature for 30 min. The cells were then permeabilized with 0.5% Triton X-100 in PBS for 15 min at room temperature. After three washes with PBS, the cells were blocked with 10% FBS at 37°C for 30 min and then incubated with a mouse monoclonal to antibody against SIRT1 (ab110304, abcam, MA) at a 1:100 dilution overnight at 4°C. The cells were then incubated with an Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L) antibody (A21202, Invitrogen, CA) at a 1:100 dilution for 1 h at 37°C. The samples were subsequently incubated with a rabbit polyclonal antibody against PGC-1 alpha (ab54481, abcam, MA) at a 1:100 dilution overnight at 4°C. Lastly, the cells were then incubated with an Alexa Fluor 647 goat anti-rabbit IgG (H+L) antibody (A21244, Invitrogen, CA) at a 1:100 dilution for 1 h at 37°C, and were imaged using a Zeiss confocal laser scanning microscope.

Guo P., Pi H., Xu S., Zhang L., Li Y., Li M., Cao Z., Tian L., Xie J., Li R., He M., Lu Y., Liu C., Duan W., Yu Z, & Zhou Z. (2014). Melatonin Improves Mitochondrial Function by Promoting MT1/SIRT1/PGC-1 Alpha-Dependent Mitochondrial Biogenesis in Cadmium-Induced Hepatotoxicity In Vitro. Toxicological Sciences, 142(1), 182-195.

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Klotho Protein Immunohistochemistry in Mouse Brain

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Mouse brains were processed for paraffin embedding, cut into 4 µm-thick sagittal sections, deparaffinized in Histoclear (National Diagnostics), and rehydrated in decreasing percentages of ethanol diluted in water. Prior to staining, antigen retrieval was performed by incubating sections in Tris EDTA pH9 buffer in a steamer for 20 min and cooling to room temperature. Sections were washed with PBS and blocked with 5% Normal Goat Serum and 0.3% Tween20 in PBS-BSA 0.1% for 30 min. Rabbit anti-mouse α-Klotho antibody (Invitrogen, #MA5-32784) was diluted 1:50 in blocking solution, added to sections, and incubated overnight at 4 °C. Slides were washed with PBS and incubated with secondary goat anti-rabbit 647 antibody (Invitrogen, A-21244, RRID: AB_2535812) for 1 h at room temperature in the dark. Slides were washed and mounted in ProLong Gold Antifade mountant with DAPI (Invitrogen, P36935).

Zhu Y., Prata L.G., Gerdes E.O., Netto J.M., Pirtskhalava T., Giorgadze N., Tripathi U., Inman C.L., Johnson K.O., Xue A., Palmer A.K., Chen T., Schaefer K., Justice J.N., Nambiar A.M., Musi N., Kritchevsky S.B., Chen J., Khosla S., Jurk D., Schafer M.J., Tchkonia T, & Kirkland J.L. (2022). Orally-active, clinically-translatable senolytics restore α-Klotho in mice and humans. EBioMedicine, 77, 103912.

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