Rat anti ctip2

Manufactured by Abcam

Sourced in United Kingdom, United States

Rat anti-CTIP2 is a primary antibody that recognizes the CTIP2 (also known as BCL11B) protein. CTIP2 is a transcriptional regulator involved in neuronal and lymphocyte development. This antibody can be used for applications such as Western blotting and immunohistochemistry.

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Close spelling variants of this product

Ctip2 (30 citations) Anti-Ctip2 (22 citations) Rat anti-Ctip2 antibody (3 citations) Rat anti-Ctip2 1/600 (2 citations)

30 protocols using rat anti ctip2

Rapid Fixation of Brain Slices

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In order to achieve rapid fixation, 300 µm acute brain slices from postnatal day (P)18–21 Nestin-spCre control and eB3 MADM-11 mice were cut, maintained for two hours in artificial cerebrospinal fluid (ACSF), then fixed overnight at 4 ˚C by rapid submersion in freshly prepared 4% PFA. Brain sections were then washed three times in 1X PBS and submerged in 30% Sucrose/PBS overnight. The sections were then re-sectioned at 40 µm. For immunostaining, sections were permeabilized for 25 min in 0.5% Triton X-100, then blocked overnight at 4 ˚C in blocking solution containing 10% FBS, 1% BSA, and 0.2% Triton X-100. The following day, sections were incubated with primary antibodies in blocking solution overnight at 4 ˚C. The following day, sections were incubated for 2 hr at room temperature with the appropriate secondary antibodies. The primary antibodies used were as follows: chicken anti-GFP (1:500, Abcam), rabbit anti-RFP (1:500, Rockland antibodies), rat anti-CTIP2 (1:500, Abcam), mouse anti-Satb2 (1:500, Abcam). The secondary antibodies used were as follows: donkey anti-chicken dylight 488 (1:500, Abcam), donkey anti-rabbit Alexafluor 594 (1:500, Jackson Immunoresearch), donkey anti-rat Alexafluor 647 (1:500, Jackson Immunoresearch), and goat anti-mouse ATTO 425 (1:250, Rockland antibodies).

Henderson N.T., Le Marchand S.J., Hruska M., Hippenmeyer S., Luo L, & Dalva M.B. (2019). Ephrin-B3 controls excitatory synapse density through cell-cell competition for EphBs. eLife, 8, e41563.

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Immunohistochemistry of Mouse Motor Cortex

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Animals were anesthetized with Avertin (0.02ml/g) and then transcardially perfused with phosphate-buffered saline (PBS) and 4% paraformaldehyde (PFA). The brain was removed and post-fixed in 4% PFA/PBS for 2 hours. The brain was sectioned coronally into 100 μm thick slices using a vibratome (VT-1000, Leica). Free-floating sections underwent antigen retrieval using L.A.B. solution (Polysciences) and were blocked in 1% BSA with 0.3% triton X-100 in PBS for 1h at room temperature. Sections were incubated with primary antibodies overnight at 4°C and then with secondary antibodies either for 2 hours in room temperature. Washes after the primary and secondary antibody were done in PBS and antibodies were diluted in PBS containing 0.2% BSA. Slices from motor cortex were mounted in PermaFluor mounting medium (Thermo Scientific) and tiled z stack images were obtained using a laser scanning confocal microscope (Zeiss LSM510).
The following primary antibodies were used: rabbit anti-dsRed2 (1:1,000, Clontech) and rat anti-CTIP2 (1:250, Abcam). The following secondary antibodies were used: Alexa Fluor 568 goat anti-rabbit (1:500 Thermo Fisher Scientific) and Alexa Fluor 647 goat anti-rat (1:500 Thermo Fisher Scientific).

Roth R.H., Cudmore R.H., Tan H.L., Hong I., Zhang Y, & Huganir R.L. (2019). Cortical Synaptic AMPA Receptor Plasticity During Motor Learning. Neuron, 105(5), 895-908.e5.

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Immunohistochemistry and In Situ Hybridization

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Brains were fixed and stained using standard methods (Galazo et al., 2016 (link); Greig et al., 2016 (link); Woodworth et al., 2016 (link)). Primary antibodies and dilutions used: rat anti-CTIP2, 1:500 (Abcam); chicken anti-GFP, 1:500 (Invitrogen); rabbit anti-GFP, 1:500 (Invitrogen); rabbit anti-RFP 1:500 (Rockland Immunochemicals), mouse anti-SATB2, 1:500 (Abcam). In situ hybridization was performed as previously described(Galazo et al., 2016 (link); Greig et al., 2016 (link); Woodworth et al., 2016 (link)). cDNA clones for riboprobes are listed in Table S4. Fluorescence in situ hybridization (FISH) for Cry-mu using DIG-labeled probes was performed as previously described(Molyneaux et al., 2009 (link)).

Sahni V., Shnider S.J., Jabaudon D., Song J.H., Itoh Y., Greig L.C, & Macklis J.D. (2021). Corticospinal neuron subpopulation-specific developmental genes prospectively indicate mature segmentally specific axon projection targeting. Cell reports, 37(3), 109843.

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Comprehensive Immunostaining and In Situ Hybridization

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Immunostaining and in situ hybridization were performed as previously described [47 (link)]. The Fezf2 cRNA probe corresponds to nucleotides 644 to 1,374 of mouse Fezf2 (GenBank: NC_000080). LacZ staining was preformed according to standard protocols. Primary antibodies used: Chicken anti β-gal (Abcam, 1:500), Rabbit anti BLBP (Millipore, 1:500), Rat anti CTIP2 (Abcam, 1:1,000), Guinea Pig anti GFAP (Advaned Immuno Chemical, 1:100), Rabbit anti PAX6 (Covance, 1:100), PHH3 (Cell Signaling, 1:100), Rabbit anti SATB2 (Abcam, 1:1,000), Goat anti SOX2 (Santa Cruz Biotech, 1:500), Rabbit anti SOX5 (Abcam, 1:500), Rabbit anti TBR1 (Abcam, 1:1,000), Mouse anti Tuj1 (Covance, 1:1,000). Primary antibodies were detected using AlexaFluor-conjugated secondary antibodies (Invitrogen, 1:1,000). DNA was visualized with DAPI (1:50,000).

Eckler M.J., Larkin K.A., McKenna W.L., Katzman S., Guo C., Roque R., Visel A., Rubenstein J.L, & Chen B. (2014). Multiple conserved regulatory domains promote Fezf2 expression in the developing cerebral cortex. Neural Development, 9, 6.

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Immunofluorescent Staining of Neuronal Markers

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At 100 DIV, cells were fixed in 4 % (wt/vol) paraformaldehyde (PFA) for 10 min before permeabilization with 0.3 % (vol/vol) Triton-X-100 in PBS for 10 min and blocking with 5 % BSA in PBS for 30 min at RT. After blocking, cells were incubated with rat anti-Ctip2 (1:300; Abcam) and mouse anti-Tuj1 (1:1000; Promega) primary antibodies diluted in the blocking solution overnight at 4°C in a humid chamber. The following day, primary antibodies were removed, and cells were washed with PBS three times for 5 min. After the last wash, the cells were incubated with Alexa Fluor 488 anti-rat and Alexa Fluor 647 anti-mouse (1:1000) secondary antibodies diluted in a blocking buffer for 1h at RT protected from light. Secondary antibodies were then removed, and cells were washed twice with PBS and then counterstained with diamidino-2- phenylindole (DAPI) (Thermo Fisher) at 0.1 μg/ml for 10min at RT and washed three times with PBS. Cells were mounted on microscope glass slides (Thermo Fisher) with ProLong^™ Diamond Antifade Mountant (Thermo Fisher). For high content imaging, plates were stored in PBS. Stained cells were imaged using a Zeiss LSM 800 microscope and analysed using Fiji software or for quantification, cells were imaged on the Opera Phenix High Content Screening System (Figure 6E).

Pantazis C.B., Yang A., Lara E., McDonough J.A., Blauwendraat C., Peng L., Oguro H., Kanaujiya J., Zou J., Sebesta D., Pratt G., Cross E., Blockwick J., Buxton P., Kinner-Bibeau L., Medura C., Tompkins C., Hughes S., Santiana M., Faghri F., Nalls M.A., Vitale D., Ballard S., Qi Y.A., Ramos D.M., Anderson K.M., Stadler J., Narayan P., Papademetriou J., Reilly L., Nelson M.P., Aggarwal S., Rosen L.U., Kirwan P., Pisupati V., Coon S.L., Scholz S.W., Priebe T., Öttl M., Dong J., Meijer M., Janssen L.J., Lourenco V.S., van der Kant R., Crusius D., Paquet D., Raulin A.C., Bu G., Held A., Wainger B.J., Gabriele R.M., Casey J.M., Wray S., Abu-Bonsrah D., Parish C.L., Beccari M.S., Cleveland D.W., Li E., Rose I.V., Kampmann M., Aristoy C.C., Verstreken P., Heinrich L., Chen M.Y., Schüle B., Dou D., Holzbaur E.L., Zanellati M.C., Basundra R., Deshmukh M., Cohen S., Khanna R., Raman M., Nevin Z.S., Matia M., Van Lent J., Timmerman V., Conklin B.R., Chase K.J., Zhang K., Funes S., Bosco D.A., Erlebach L., Welzer M., Kronenberg-Versteeg D., Lyu G., Arenas E., Coccia E., Sarrafha L., Ahfeldt T., Marioni J.C., Skarnes W.C., Cookson M.R., Ward M.E, & Merkle F.T. (2022). A reference human induced pluripotent stem cell line for large-scale collaborative studies. Cell stem cell, 29(12), 1685-1702.e22.

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Immunohistochemical Analysis of Embryonic and Postnatal Mouse Brain

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Embryos were dissected, and the brains were fixed in 4% paraformaldehyde (PFA) for 1–3.5 hr. For postnatal stage, brains were fixed with 4%PFA overnight. Following 30% sucrose replacement, fixed brains were embedded in optimum cutting temperature (OCT) compound, and 20 micrometer slices were cut on a cryostat. The antibodies used were, rat anti-GFP (1∶500; nakalai tesk), rabbit anti-GFP (1∶200; IBL), rabbit anti-DsRed (1∶500; Invitrogen), goat anti-Unc5D (1∶100; R&D), rabbit anti-Tbr2 (1∶300; abcam), goat anti-NeuroD1 (1∶100; Santa Cruz), mouse anti-PCNA (1∶100; Cell Signaling), mouse anti-Tuj1 (1∶500; SIGMA), rabbit anti-Tbr1 (1∶100; abcam), mouse anti-RORb (1∶100; PERSEUS PROTEOMICS), rat anti-Ctip2 (1∶300; abcam), goat anti-Brn2 (1∶100; Santa Cruz), and mouse anti-Prdm8 [24] (link). Alexa Fluor-conjugated secondary antibodies (Invitrogen) were also used. EdU labeling (intraperitoneal injection of 12.5 mg/kg EdU) and staining were performed according to manufacturer's instructions (Invitrogen). Stainings were examined with Zeiss LSM 710 or Olympus IX81, and the images were finally processed with Adobe Photoshop.

Inoue M., Kuroda T., Honda A., Komabayashi-Suzuki M., Komai T., Shinkai Y, & Mizutani K.I. (2014). Prdm8 Regulates the Morphological Transition at Multipolar Phase during Neocortical Development. PLoS ONE, 9(1), e86356.

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Pcdh19 Antibody Generation and Validation

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Pcdh19 antibody was raised by immunizing a rabbit with the GST-fused cytoplasmic region of Pcdh19 (Val1041-Leu1140 a.a.) and subsequently affinity-purified with the antigen. A GST-reactive population was also removed. The following antibodies were purchased: rat anti-Ctip2, chicken anti-LacZ and chicken anti-GFP (Abcam); mouse anti-Homer1 and guinea pig anti-Synapsin1/2 (Synaptic systems); rabbit anti-MAP2, mouse anti-Tau1 and guinea pig anti-Vglut2 (Millipore); rabbit anti-FLAG and mouse anti-MAP2 (Sigma-Aldrich); and mouse anti-Abi-1 and rabbit anti-RFP (MBL).

Hayashi S., Inoue Y., Hattori S., Kaneko M., Shioi G., Miyakawa T, & Takeichi M. (2017). Loss of X-linked Protocadherin-19 differentially affects the behavior of heterozygous female and hemizygous male mice. Scientific Reports, 7, 5801.

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Immunohistochemistry and In Situ Hybridization

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Brain Tissue Fixation, Sectioning, and Immunostaining

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Transcardial perfusion with 4% paraformaldehyde (PFA) was performed on the mice for fixation. The brains of E17.5, P0, P2, P4, P7, P14, P30 mice were dissected and post-fixed at 4°C with 4% PFA for 2h to overnight, depending on the size of brains. The brains were dehydrated in 30% sucrose and embedded in OCT compound. Cryosections were cut at 14-μm, or 80-μm thickness with a Cryostat (HM505E, Microm, Germany). Immunostaining was performed with standard protocols: brain sections were incubated overnight with primary antibodies at 4°C and incubated with appropriate fluorescent secondary antibodies for 2h at room temperature. The following primary antibodies were used: Goat anti-GFP (1:1000, Novus Biologicals); Mouse anti-FOXP1 (1:125, Abcam); Rabbit anti-Tbr2 (1:300, Abcam); Rabbit anti-Tbr1 (1:200, Abcam); Mouse anti-Satb2 (1:100, Santa Cruz); Mouse anti-Nestin (1:200, Abcam); Rabbit anti-CDP (1:50, Santa Cruz); Rat anti-Ctip2 (1:500, Abcam); Mouse anti-phospho Histone H3(1:300, Abcam); Mouse anti-β-III-tubulin (1:500, Promega).
Immunofluorescence images were obtained using either Olympus BX41 or Zeiss LSM 710 confocal microscope. The morphology of neurons in the cortex or culture was traced and analyzed using Neurolucida software (MBF Bioscience).

Li X., Xiao J., Fröhlich H., Tu X., Li L., Xu Y., Cao H., Qu J., Rappold G.A, & Chen J.G. (2015). Foxp1 Regulates Cortical Radial Migration and Neuronal Morphogenesis in Developing Cerebral Cortex. PLoS ONE, 10(5), e0127671.

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Immunofluorescent Staining: Detailed Protocols

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For immunofluorescent staining, sections were rinsed in PBS and blocked with 5% normal serum/0.1% Triton X-100/PBS at room temperature. Primary antibodies were diluted in blocking solution and incubated 1–2 days at 4°C with gentle agitation. The antibodies utilized were; rabbit anti-Parvalbumin (Swant), chicken anti-EGFP (Aves Labs), rat anti-CTIP2 (Abcam), rabbit anti-SATB2 (Abcam), rabbit anti-CUX1 (Santa Cruz), mouse anti-NEUN (Chemicon), rabbit anti-Cleaved Caspase-3 (Cell Signaling Technology), rabbit anti-IBA1 (Wako), goat anti-IGF1 (R&D Research), rabbit anti-PKCγ (Santa Cruz), rabbit anti-MAP2K1/2(MEK1/2) (Abcam), rabbit anti-P-MAPK1/3(ERK1/2) (Cell Signaling Technology) and rabbit anti-IGF1Rβ (Cell signaling Technology). After rinsing in PBS/T, the secondary antibody was diluted in blocking solution and added overnight at 4°C. Secondary antibodies included Alexa Fluor 488, 546 or 568, and 647 conjugated anti-rabbit, anti-mouse, anti-rat, or anti-goat IgG (Invitrogen). For some experiments slides were then incubated in Hoechst or DAPI for nuclear labeling, rinsed, and mounted. Images were collected with a Zeiss LSM 710, 780, or Leica SP5 laser scanning confocal microscope.

Xing L., Larsen R.S., Bjorklund G.R., Li X., Wu Y., Philpot B.D., Snider W.D, & Newbern J.M. (2016). Layer specific and general requirements for ERK/MAPK signaling in the developing neocortex. eLife, 5, e11123.

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