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Ab5622

Manufactured by Abcam
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Sourced in United Kingdom, United States

AB5622 is a rabbit polyclonal antibody raised against a synthetic peptide corresponding to the N-terminal region of human Angiopoietin-1. This antibody is designed for use in immunochemical techniques such as Western blotting.

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Lab products found in correlation

Triton x 100, by Thermo Fisher Scientific (2 mentions) Normal donkey serum (nds), by Jackson ImmunoResearch (2 mentions) Dab substrate, by Vector Laboratories (1 mentions) Ab135711, by Abcam (1 mentions) Tissue tek, by Sakura Finetek (1 mentions) Mab1637, by Merck Group (1 mentions) Nanog, by Cell Signaling Technology (1 mentions) Ab18723, by Abcam (1 mentions) Ab18465, by Abcam (1 mentions) T brachyury, by Santa Cruz Biotechnology (1 mentions) Hnpp fluorescent detection set, by Roche (1 mentions) Hoechst 33342, by Dojindo Laboratories (1 mentions) A31573, by Thermo Fisher Scientific (1 mentions) A31571, by Thermo Fisher Scientific (1 mentions) Paraformaldehyde (pfa), by Merck Group (1 mentions) Mab2166, by Merck Group (1 mentions) A10036, by Thermo Fisher Scientific (1 mentions) A10040, by Thermo Fisher Scientific (1 mentions) Ab55152, by Abcam (1 mentions) Ab92391, by Abcam (1 mentions)

10 protocols using ab5622

1

Immunohistochemical Profiling of Synovial Thyroid Enzymes

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Of synovial tissue samples, 8 pieces of roughly 0.8 cm2 were used for immunohistochemistry. Samples were immediately placed in freezing medium (Tissue Tek, Sakura Finetek, Zoeterwoude, The Netherlands) and then quick-frozen in liquid nitrogen. Some samples were fixed in formalin and embedded in paraffin according to standard procedures.
Then, 6–8 μm sections were stained with a panel of antibodies directed against DIO1 (NPB1-19706, Novus Biologicals, via R&D Systems), DIO2 (ab135711, abcam, Cambridge, UK), DIO3 (NBP 1-05767, Novus Biologicals, via R&D Systems), MCT8 (thyroid hormone transporter, ab104689, abcam), TRα (ab42565, abcam), TRβ (ab5622, abcam), and trace amine associated receptor 1 (TA1, ab65633, abcam). Primary staining was visualized using respective secondary antibody conjugated to horseradish peroxidase (Dako, Glostrup, Denmark) and making use of the DAB substrate (Vector Laboratories, Burlingame, USA). In immunohistochemistry experiments, we controlled staining by using unspecific serum or unspecific IgG antibodies instead of primary antibodies and this constantly yielded negative result (see Figures).
The density of positive cells for DIO1, DIO2, and DIO3 was averaged from 17 randomly selected high-power fields at the magnification of 400x and expressed as cell number per square millimeter.
Pörings A.S., Lowin T., Dufner B., Grifka J, & Straub R.H. (2019). A thyroid hormone network exists in synovial fibroblasts of rheumatoid arthritis and osteoarthritis patients. Scientific Reports, 9, 13235.
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2

Immunofluorescence Staining of Stem Cells

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Cells were fixed with 4% (w/v) paraformaldehyde in PBS for 10 min, permeabilized with 0.1% (v/v) Triton X-100 in PBS for 15 min, and blocked in 1% normal donkey serum for 1 h. Cells were incubated with primary antibodies for 1 h, washed with PBS, and incubated with appropriate Alexa Fluor-conjugated secondary antibodies for 1 h. Nuclei were counterstained with 20 µM Hoechst 33342 (Dojindo, H342) for 10 min. Primary antibodies were as follows: Nanog (1∶800; Cell Signaling, 4903), Oct4 (1∶100; SantaCruz, sc-5279), Sox2 (1∶100; R&D Systems, MAB2018), SSEA-4 (1∶100; Stemgent, 09-0006), Tra-1-60 (1∶100; Stemgent, 09-0010), Tra-1-81 (1∶100; Stemgent, 09-0011), Pax6 (1∶200; BD Biosciences, 561462), Sox1 (1∶100; Stemgent, 09-0084), T/Brachyury (1∶200; SantaCruz, sc-17745), Tuj1 (1∶500; Millipore, MAB1637), MAP2 (1∶500; Millipore, AB5622), DCX (1∶500; Abcam, ab18723), and CTIP2 (1∶500; Abcam, ab18465). Alkaline phosphatase staining was performed using the HNPP Fluorescent Detection Set (Roche).
Ono T., Suzuki Y., Kato Y., Fujita R., Araki T., Yamashita T., Kato H., Torii R, & Sato N. (2014). A Single-Cell and Feeder-Free Culture System for Monkey Embryonic Stem Cells. PLoS ONE, 9(2), e88346.
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3

Immunofluorescence Staining of Cultured Cells

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Cells were fixed using 4% paraformaldehyde (Sigma, 158127) in 0.1 M PBS, pH 7.4 (Corning, 21-040-CV) for 30 min. After three washes in cold PBS, cells were permeabilized and blocked for 1 h at room temperature (RT) using 0.1% Triton X-100 (Thermo Fisher Scientific, 28313) and 4% normal donkey serum (Jackson Immuno Research, 017-000-121) in PBS. Primary antibodies were added in the presence of blocking buffer ON at 4 °C. Secondary antibodies (1:500) were added after three PBS washes in blocking buffer at RT for 1 h. The following primary antibodies were used for the immunofluorescence studies: rabbit anti-DARPP-32 (Santa Cruz, sc-271111, 1:100), rabbit anti-MAP2 (Millipore, AB5622, 1:100), rabbit anti-Nestin (Abcam, ab92391, 1:00), mouse anti-MHC-class-II (Abcam, ab55152, 1:100), rabbit anti-Cleaved Caspase-3 (CellSignal, 9661, 1:100), and mouse anti-HTT (Millipore, MAB2166, 1:100). The secondary antibodies were donkey anti-rabbit, anti-mouse IgG conjugated with Alexa-546 (Invitrogen, A10040 and A10036) or Alexa-647 (Invitrogen, A-31573 and A-31571). Images were acquired using a Biotek Cytation 5 microscope and were prepared using Fiji software (ImageJ, https://fiji.sc/).
Tshilenge K.T., Aguirre C.G., Bons J., Gerencser A.A., Basisty N., Song S., Rose J., Lopez-Ramirez A., Naphade S., Loureiro A., Battistoni E., Milani M., Wehrfritz C., Holtz A., Hetz C., Mooney S.D., Schilling B, & Ellerby L.M. (2023). Proteomic Analysis of Huntington’s Disease Medium Spiny Neurons Identifies Alterations in Lipid Droplets. Molecular & Cellular Proteomics : MCP, 22(5), 100534.
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4

Immunofluorescence Staining of Neuronal Markers

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C116 and HD MSN were fixed using 4% paraformaldehyde in 0.1 M PBS, pH 7.4 (Corning, 21–040-CV) for 30 min. After three washes in PBS, cells were permeabilized and blocked for 1 h at RT using 0.1% Triton X-100 (Thermo Fisher Scientific, 28,313) and 4% donkey serum in PBS. Primary antibodies were added in the presence of blocking buffer ON at 4°C. Secondary antibodies (1:500) were added after three PBS washes in blocking buffer at RT for 1 h. The following primary antibodies were used for the immunofluorescence studies: rabbit anti-PPP1R1B/DARPP-32 (1:100; Santa Cruz Biotechnology, sc-11,365), rabbit anti-MAP2 (1:100; Millipore, AB5622) and rabbit anti-NES/nestin (1:100; Abcam, ab92391). The secondary antibodies were donkey anti-rabbit IgG conjugated with Alexa Fluor 488 (Invitrogen, A12379) or Alexa Fluor 647 (Invitrogen, A22287). Images were acquired using a Biotek Cytation 5 microscope and were prepared using Fiji software (ImageJ).
Bailus B.J., Scheeler S.M., Simons J., Sanchez M.A., Tshilenge K.T., Creus-Muncunill J., Naphade S., Lopez-Ramirez A., Zhang N., Lakshika Madushani K., Moroz S., Loureiro A., Schreiber K.H., Hausch F., Kennedy B.K., Ehrlich M.E, & Ellerby L.M. (2021). Modulating FKBP5/FKBP51 and autophagy lowers HTT (huntingtin) levels. Autophagy, 17(12), 4119-4140.
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5

Immunoprecipitation of TRβ1 and ACAA2 Detection

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Heart tissue was homogenized in cold lysis buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) with protease inhibitor mixture. 300 μg of heart lysates were precleared with protein A/G beads (Roche Molecular Biochemicals) and incubated with TRβ1 antibody MA1-216 (Thermo Fisher, Waltham, MA) or AB5622 (Abcam, Cambridge, MA) and protein A/G-agarose beads overnight. Negative controls reactions used isotype IgGs. Complexed proteins were recovered by boiling beads in SDS sample buffer, standard SDS-PAGE and immunoblotting for ACAA2, (#GTX115417, GeneTex, Irvine, CA), was performed.
Wang W, & Ledee D. (2021). ACAA2 is a Ligand-dependent Coactivator for Thyroid Hormone Receptor β1. Biochemical and biophysical research communications, 576, 15-21.
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6

Immunofluorescence Staining of Neural Markers

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MN cultures were washed with warm 1X PBS and immediately fixed in 4% PFA for 15 minutes. Samples were permeabilized in 0.5% triton for 10 minutes and blocked in blocking solution (1 mg/mL BSA; 10% Goat serum; 0.1% triton in PBS) for 1 hour at room temperature. Samples were incubated with primary antibodies in blocking solution for 12 hours at 4 °C. Antibodies were used in the following concentrations: Elavl2 1:50 (Rabbit; Proteintech, 14008-1-AP), NFH 1:500 (Chicken; Abcam, ab72996) 1:1,000 MAP2 (Rabbit; Millipore, AB5622), 1:100 Tau (Mouse; Abcam, ab80569). Samples were then incubated for 2 hours with fluorescent secondary antibodies: DyLight 405 anti-chicken 1:200; Alexafluor 488 anti-rabbit 1:500; AlexaFluor 647 anti-mouse 1:500; AlexaFluor 594 anti-rabbit 1:500. Samples were mounted using ProLong® gold antifade reagent.
Rotem N., Magen I., Ionescu A., Gershoni-Emek N., Altman T., Costa C.J., Gradus T., Pasmanik-Chor M., Willis D.E., Ben-Dov I.Z., Hornstein E, & Perlson E. (2017). ALS Along the Axons – Expression of Coding and Noncoding RNA Differs in Axons of ALS models. Scientific Reports, 7, 44500.
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7

Immunocytochemical Profiling of Stem Cells

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Cells were fixed with 4% PFA, permeabilized and blocked with 0.3% Triton X-100, 6% bovine serum in the TBS buffer. Samples were incubated overnight at 4ºC with the primary antibodies against SEEA4 (Thermo, MA1-021-D488, 1:100), SOX2 (Thermo, MA1-014-D488, 1:100); Nestin (BioLegend, 656812, 1:100); SOX1 (Abcam, ab8775, 1:200); MAP2 (Merck Millipore, AB5622, 1:200), and TUJ1 (Abcam, ab224978, 1:200), respectively. Alexa-Fluor-Dye secondary antibodies were applied (1:500) at room temperature for 1 h. After staining, cells were washed twice and dried. Stained cells were photographed and images were acquired under a microscope of Tissue FAXS i4 SCAN (Zeiss, Dublin, CA, USA).
Wu Y.T., Tay H.Y., Yang J.T., Liao H.H., Ma Y.S, & Wei Y.H. (2023). Mitochondrial impairment and synaptic dysfunction are associated with neurological defects in iPSCs-derived cortical neurons of MERRF patients. Journal of Biomedical Science, 30, 70.
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8

Immunofluorescence Staining of Mouse Brain Tissue

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Mice were sacrificed 7 and 35 days post‐dMCAO. Coronal slices (25‐μm‐thick) were prepared for immunofluorescence staining as previously described.18 The following primary antibodies were used: rabbit anti‐NeuN (EMD Millipore), rabbit anti‐microtubule‐associated protein 2 (MAP2; sc‐20,172; Santa Cruz), mouse anti‐200kD neurofilament heavy (NF200; MAB5262; Millipore Sigma), rabbit anti‐beta‐amyloid precursor protein (β‐APP; 512700; Fisher Scientific), rabbit anti‐myelin basic protein (MBP; ab5622; Abcam), goat anti‐Iba1 (ab5076; Abcam), goat anti‐CD206 (AF2535; R&D Systems), rat anti‐CD16 (553142; BD Pharmingen), and mouse anti‐SMI‐32 (ab50761; Abcam). Samples were blocked in 5% normal donkey serum (Jackson ImmunoResearch) to reduce nonspecific binding. When a mouse primary antibody was used, a mouse‐on‐mouse (M.O.M) blocking reagent (MKB‐2213; Vector Laboratories) was used to avoid cross‐reactivity. Donkey Cy3‐ or DyLight 488‐conjugated secondary antibodies (Jackson ImmunoResearch) were used. Images were obtained using an Olympus FluoView FV1000 confocal microscope (Olympus America) and analyzed with ImageJ. Cell numbers were calculated from two random microscopic fields in each mouse brain.
Liu L., Liu J., Li M., Lyu J., Su W., Feng S, & Ji X. (2022). Selective brain hypothermia attenuates focal cerebral ischemic injury and improves long‐term neurological outcome in aged female mice. CNS Neuroscience & Therapeutics, 29(1), 129-139.
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9

Per2 Promoter Chromatin Immunoprecipitation

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DNA samples were obtained using SimpleChIP® Enzymatic Chromatin IP Kit (9002, CST, United States) according to the instructions, and anti-thyroid hormone receptor beta (ab5622, Abcam, United States, 1:50, RRID:AB_304991) antibody was used for immunoprecipitation. Per2 promoter specific primers: Primer1: forward 5′-AGTCGCCGCTGTCACATAG-3′, reverse 5′-AGGAACCGACGAGGTGAAC-3′; Perimer2: forward 5′-AAGTTGTCCTTGTCTCACC-3′, reverse 5′-AGG AACCGACGAGGTGAAC-3′; Primer3: forward 5′-GGGTG TCCACTCTTTGGT-3′, reverse 5′-CCTGGTCTATGTCTGG GA-3′.
Chen X., Wu M., Liang N., Lu J., Qu S, & Chen H. (2021). Thyroid Hormone-Regulated Expression of Period2 Promotes Liver Urate Production. Frontiers in Cell and Developmental Biology, 9, 636802.
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10

Dendritic Complexity Analysis in Neuronal Cultures

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Neuronal primary cultures from WT and KO animals were treated with 0.2 mM SB216763, and the complexity of dendritic arborization and connectivity was analyzed. After 7 DIV, the cell culture was fixed and immunostained with Rb-Map2 antibody (ab5622; Abcam) (following the immunofluorescence staining protocol described above). Pictures and z stacks were obtained using a Zeiss Axio Observer Z1 microscope at 103 magnification with acquisition software ZEN 2011. The images were processed using ImageJ Fiji version 1.50b (http:// imagej.nih.gov) using Auto-Threshold (algorithms Mean). Maximum-intensity projection was used for the reconstruction of the three-dimensional dendritic network using NeuronStudio software version 0.9.92. Automated segmentation was obtained with picture thresholding and Sholl analysis taking as a starting point a soma and counting over a length of 20 mm with concentric circles of 10 mm (Sholl, 1953) .
Jorge-Torres O.C., Szczesna K., Roa L., Casal C., Gonzalez-Somermeyer L., Soler M., Velasco C.D., Martínez-San Segundo P., Petazzi P., Sáez M.A., Delgado-Morales R., Fourcade S., Pujol A., Huertas D., Llobet A., Guil S, & Esteller M. (2018). Inhibition of Gsk3b Reduces Nfkb1 Signaling and Rescues Synaptic Activity to Improve the Rett Syndrome Phenotype in Mecp2-Knockout Mice. Cell reports, 23(6).
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