Ab28448

Manufactured by Abcam

Sourced in United Kingdom

Ab28448 is a recombinant anti-CD3 antibody. It is designed for use in various immunological applications.

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Lab products found in correlation

6 protocols using ab28448

Immunofluorescence Staining of Neural Cells

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For immunofluorescence staining, cells were fixed with 4% formaldehyde in PBS solution for 15 min, permeabilized with 1% Triton X-100 in PBS solution for 15 min and blocked with 2% bovine serum albumin in PBS solution for 1 h. Thereafter, cells were incubated with the following primary antibodies overnight at 4 °C: anti-MAP 2 (1:500; Sigma, M1406), anti-TUJ1 (1:500; Covance, MRB-435P), anti-TAU (1:200; Dako, A0024) and anti-CTIP2 (1:200; Abcam, ab28448). Primary antibodies were detected using secondary antibodies conjugated to Alexa Fluor 488 (1:500; Molecular Probes) and Alexa Fluor 594 (1:500; Molecular Probes). Cells were washed in PBS and nuclei counterstained with DAPI (Sigma-Aldrich, D9542). Finally, coverslips of growing cells were transferred onto glass slides with mounting medium (Dako, S3023) and imaged immediately using sequential line scanning with a Leica TCS SP5 II inverted confocal microscope.

Aldana B.I., Zhang Y., Jensen P., Chandrasekaran A., Christensen S.K., Nielsen T.T., Nielsen J.E., Hyttel P., Larsen M.R., Waagepetersen H.S, & Freude K.K. (2020). Glutamate-glutamine homeostasis is perturbed in neurons and astrocytes derived from patient iPSC models of frontotemporal dementia. Molecular Brain, 13, 125.

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Immunofluorescent labeling of neural markers

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The primary antibodies were rabbit TRIM32 antibody (sc-99011, Santa Cruz, Dallas, TX, USA), mouse TRIM32 antibody (SAB1407164, Sigma, St. Louis, MO, USA), rabbit anti-TBR1 (ab3190, Abcam, Cambridge, UK), rabbit anti- BCL11B (ab28448, Abcam, Cambridge, UK), rabbit anti-CUX1 (sc-13024, Santa Cruz, Dallas, TX, USA), chicken anti-PAX6 (AB_528427, Developmental Studies Hybridoma Bank, Lowa, IA, USA), rabbit anti-TBR2 (ab23345, Abcam, Cambridge, UK), Click-iT™ EdU imaging kit (C10086, Thermo Fisher, Rockford, IL, USA), rabbit anti-PH3 (9713P, Cell Signaling Technology, Boston, MA, USA), rabbit anti-active caspase-3 (C8487, Sigma, St. Louis, MO, USA). The corresponding secondary antibodies conjugated with Alexa fluorophores 488/555/647 (A21202, A31570, A28181, A21206, A31572, A32795, A11039) were from Invitrogen.

Sun Y.Y., Chen W.J., Huang Z.P., Yang G., Wu M.L., Xu D.E., Yang W.L., Luo Y.C., Xiao Z.C., Xu R.X, & Ma Q.H. (2022). TRIM32 Deficiency Impairs the Generation of Pyramidal Neurons in Developing Cerebral Cortex. Cells, 11(3), 449.

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Probing CTIP2 and CD45 in Aorta

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BCL11B/CTIP2 expression in the aorta was probed using five antibodies that recognize different epitopes on CTIP2: anti-CTIP2 rat monoclonal (Abcam, ab18465), rabbit polyclonal (Abcam, ab70452), rabbit polyclonal (Abcam, ab28448), rabbit monoclonal (Abcam, ab187668), and rabbit monoclonal (CST signaling 12120). Western blot and IHC staining antibody dilutions were used as recommended by the manufacturer. CD45 expression in the aorta was probed with anti-CD45 rabbit polyclonal (Abcam, ab10558) at 1:2000 dilution. Beta-actin rabbit polyclonal immunoglobulin G (ThermoFisher Scientific) was used for the western blot loading control at 1:1000 dilution. Donkey anti-rabbit (LI-COR) IRDye® 800CW and goat anti-rat (ThermoFisher Scientific) Alexa Fluor® 680-conjugated secondary antibodies were used at 1:5000 dilution for western blot.

Maskari R.A., Hardege I., Cleary S., Figg N., Li Y., Siew K., Khir A., Yu Y., Liu P., Wilkinson I., O’Shaughnessy K, & Y.a.s.m.i.n. (2018). Functional characterization of common BCL11B gene desert variants suggests a lymphocyte-mediated association of BCL11B with aortic stiffness. European Journal of Human Genetics, 26(11), 1648-1657.

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Protein Extraction and Western Blotting for Ctip2 Analysis

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Total protein extract was isolated from human tissue using TRI Reagent (T9424, Sigma-Aldrich, Madrid, Spain) according to the manufacturer’s protocol. Total protein extracts were denatured using 2.5 mmol/l dithiothreitol at 100 °C for 5 minutes and then 40 μg of each denatured samples was subjected to 12% SDS-PAGE and transferred to a nitrocellulose membrane (IPVH00010, Millipore, Barcelona, Spain) for 90 minutes at 1 mA/cm² as described previously.^{38 (link)} Membranes were incubated with polyclonal rabbit anti-Ctip2 (overnight at +4 °C, dilution of 1 µg/ml; Abcam Ab28448) and anti-Actin (20 minutes at RT, dilution of 1:20,000, MP Biochemicals), diluted in immunoblot buffer (Tris-buffered saline (TBS) containing 0.05% Tween-20 and 5% no-fat dry milk).

Straccia M., Garcia-Diaz Barriga G., Sanders P., Bombau G., Carrere J., Mairal P.B., Vinh N.N., Yung S., Kelly C.M., Svendsen C.N., Kemp P.J., Arjomand J., Schoenfeld R.C., Alberch J., Allen N.D., Rosser A.E, & Canals J.M. (2015). Quantitative high-throughput gene expression profiling of human striatal development to screen stem cell–derived medium spiny neurons. Molecular Therapy. Methods & Clinical Development, 2, 15030-.

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Aortic BCL11B/CTIP2 and CD45 Expression

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BCL11B/CTIP2 expression in the aorta was probed using 5 antibodies that
recognize different epitopes on CTIP2: Anti-CTIP2 rat monoclonal (Abcam,
ab18465), rabbit polyclonal (Abcam, ab70452), rabbit polyclonal (Abcam,
ab28448), rabbit monoclonal (Abcam, ab187668) and rabbit monoclonal (CST
signaling 12120). Western blot and immunohistochemical staining antibody
dilutions were used as recommended by the manufacturer. CD45 expression in the
aorta was probed with anti-CD45 rabbit polyclonal (Abcam, ab10558) at 1:2000
dilution. Beta-actin rabbit polyclonal IgG (ThermoFisher Scientific) was used
for the western blot loading control at 1:1000 dilution. Donkey anti-rabbit
(Li-Cor) IRDye® 800CW and goat anti-rat (ThermoFisher Scientific) Alexa
Fluor® 680 conjugated secondary antibodies were used at 1:5000 dilution
for western blot.

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Immunofluorescence Staining for Neural Markers

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Slices were used for detection and quantification of Ctip-2, NeuN, Sox-2, Ki67, S100-β, GFAP, or Iba-1 immunofluorescence. After preincubation for 1 h with Tris-buffered saline with 0.1% Triton X-100 (pH 7.5), sections were sequentially incubated overnight at 4ºC with the following polyclonal antibodies: (i) anti-Ctip-2 (ref. ab28448, Abcam, Cambridge, UK) used at 1:400; (ii) anti-NeuN (ref. ABN78, Millipore, MA, USA) used at 1:100; (iii) anti-Sox-2 (ref. EPR3131, Abcam, Cambridge, UK) used at 1:200; (iv) anti-S100-β (ref. ab868, Abcam, Cambridge, UK) used at 1:500; (v) anti-Iba-1 (ref. 019-19741, Wako Chemicals, Richmond, VI, USA) used at 1:500; (vi) anti-GFAP (ref. Z0334, Dako Cytomation, Glostrup, Denmark) used at 1:200; or (vii) anti-Ki67 (ref. ab833, Abcam, Cambridge, UK) used at 1:100, followed by washing in Tris-buffered saline and a new incubation (at 37ºC for 2 h) with an anti-rabbit or an anti-mouse, as required, secondary antibody conjugated with Alexa 488 or 546 (Invitrogen, Carlsbad, CA, USA). A DMRB microscope and a DMC4500Fx camera (Leica, Wetzlar, Germany) were used for slide observation and photography.

Santos-García I., Rodríguez-Cueto C., Villegas P., Piscitelli F., Lauritano A., Shen C.K., Di Marzo V., Fernández-Ruiz J, & de Lago E. (2023). Preclinical investigation in FAAH inhibition as a neuroprotective therapy for frontotemporal dementia using TDP-43 transgenic male mice. Journal of Neuroinflammation, 20, 108.

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