Immunohistochemical labeling of embryonic/postnatal brain sections or isolated cortical cells was performed as described earlier (Higginbotham et al., 2013 (link); Yokota et al., 2009 (link)). The following primary antibodies were used: acetylated tubulin (mouse, T6793, Sigma-Aldrich), α-tubulin (mouse, T6199, Sigma-Aldrich), BLBP (rabbit, ab32423, Abcam), BrdU (mouse, 347580, Becton Dickinson), Ctip2 (rat, ab18465, Abcam), Cux1 (rabbit, sc-13024, Santa Cruz Biotechnology), GABA (rabbit, A2052, Sigma-Aldrich), GFAP (rabbit, Z0334, DAKO), GFP (chicken, ab13970, Abcam), laminin (rabbit, L9393, Sigma), MEMO1 (rabbit, ab124901, Abcam), NeuN (mouse, MAB377, Chemicon), phospho-Vimentin (mouse, ab22651, Abcam), Pou3f2 (mouse, sc-393324, Santa Cruz Biotechnology), PTPRZ1 (rabbit, HPA015103, Sigma-Aldrich), PV (mouse, MAB1572, Millipore), RC2 (mouse IgM, Developmental Studies Hybridoma Bank, University of Iowa), RFP (rabbit, 600-401-379, Rockland), SST (rat, MAB354, Millipore), Tbr1 (rabbit, ab31940, Abcam), Tbr2 (rat, 14-4875, eBioscience), Tuj1 (mouse, 1409, STEMCELL technologies) and tyrosinated tubulin (rat, ab6160, Abcam). Appropriate Cy2, Cy3 or Alexa dye-conjugated secondary antibodies (Jackson ImmunoResearch, Molecular Probes) were used to detect primary antibody binding. DAPI (Sigma-Aldrich, D9542) was used as a nuclear counterstain.
Ab22651
Manufactured by Abcam
Ab22651 is a primary antibody product manufactured by Abcam. It is designed for use in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab31940, by Abcam (2 mentions)
Ab18465, by Abcam (2 mentions)
Hpa015103, by Merck Group (2 mentions)
Ab18259, by Abcam (2 mentions)
Ab13970, by Abcam (1 mentions)
Mab377, by Merck Group (1 mentions)
Mab354, by Merck Group (1 mentions)
Mab1572, by Merck Group (1 mentions)
A2052, by Merck Group (1 mentions)
Z0334, by Agilent Technologies (1 mentions)
T6793, by Merck Group (1 mentions)
L9393, by Merck Group (1 mentions)
T6199, by Merck Group (1 mentions)
Ab6160, by Abcam (1 mentions)
Ab32423, by Abcam (1 mentions)
Ab124901, by Abcam (1 mentions)
Sc 13024, by Santa Cruz Biotechnology (1 mentions)
Sc 393324, by Santa Cruz Biotechnology (1 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (1 mentions)
Ab14955, by Abcam (1 mentions)
5 protocols using ab22651
1
Immunohistochemical Labeling of Brain
2
Immunohistochemical analysis of neural progenitor cells
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Upon completion of timelapse imaging, brain slices were fixed in 4% PFA in PBS overnight at 4 °C and then transferred to PBS. Slices were subjected to boiling citrate-based antigen retrieval solution for 20 min and permeabilized and blocked in blocking buffer (PBS plus 0.1% Triton X-100, 10% serum, and 0.2% gelatin) for 1 h. Primary antibodies were diluted in blocking buffer and applied to slices for 36 h at 4 °C. Slices were washed with PBS plus 0.5% Triton X-100 and then incubated in secondary antibodies diluted in blocking buffer for 3–5 h. Images were acquired on a Leica TCS SP5 X laser confocal microscope. Primary and secondary antibodies used: goat anti-SOX2 (Santa Cruz Biotechnology, sc-17320, 1:250), rabbit anti-TBR2 (Abcam, ab23345, 1:100), chicken anti-GFP (Aves Labs, GFP-1020, 1:1,000), rabbit anti-FOXG1 (Abcam, ab18259, 1:250), mouse anti-pVIM (Abcam, ab22651, 1:250), mouse anti-phosphohistoneH3 (Abcam, ab14955, 1:200), AlexaFluor 488, 546, 594, or 647-conjugated donkey anti-goat, -rabbit, -mouse IgG (Invitrogen, 1:500), and AlexaFluor 488 donkey anti-chicken IgY (Jackson ImmunoResearch, 703-545-155, 1:500).
3
Western Blot Analysis of Vimentin and β-catenin
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Cells were lysed in RIPA buffer (Beyotime) on ice for 5 min to collect total protein. After protein concentration examination by a bicinchoninic acid kit (Thermo Fisher Scientific), an equal amount of protein sample (30 µg) was run on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and loaded onto polyvinylidene fluoride membranes. The membranes were treated with nonfat milk at 20°C for 2 h and then hybridized with the primary antibodies (all diluted at 1:1,000) against Vimentin (ab22651, Abcam), β-catenin (ab231305, Abcam) and GAPDH (ab8245, Abcam) for 12 h at 4°C, and then hybridized with the secondary antibody (1:10,000, #7076, CST) for 1 h at 20°C. The protein bands were developed using the enhanced chemiluminescence kit (Thermo Fisher Scientific), and the protein quantification was conducted using an Image-Pro® Plus Software. GAPDH was used as the endogenous reference [25 (link)].
4
Shear stress-induced Jagged1 and vimentin signaling
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For shear stress experiments with imaging as readout, HUVEC were seeded into collagen IV coated 6-channel slides from ibidi (μ-slide VI 0.4, #80606) at 106 cells/ml. The next day HUVEC were exposed to 1 Pa shear stress for 24 hours or kept as a static control. Afterwards the cells were washed twice with PBS and fixed in 4% PFA for 20 minutes. Fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 15 minutes and blocked with 4% donkey serum in PBS for 30 minutes. The cells were stained for Jagged1 (H-66, Santa Cruz) and S55 phospho-vimentin (ab22651, Abcam) overnight at 4 °C. Secondary antibodies (A21206 and A21146, ThermoFisher) were incubated for 45 minutes at RT. The nuclei were stained with DAPI (Sigma) for 5 minutes. After a final washing step with PBS Mowiol was injected into the channels as a mounting medium. The cells were imaged with a Zeiss Axiovert 200 M, using a 40x/0.95 objective.
5
Comprehensive Immunohistochemical Antibody Panel
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The antibodies to EMX2 (ab94713; 1:50), FOXG1 (ab18259; 1:400), p-VIM (ab22651; 1:120), SOX2 (ab79351; 1:500), SATB2 (ab51502; 1:50), CTIP2 (ab18465; 1:250), TBR1 (ab31940; 1:500) and CUX1 (ab54583; 1:200) were from Abcam. The antibodies to OCT3/4 (sc5279; 1:22) and LIFR (sc-515337; 1:100) were from Santa Cruz. The antibody to PAX6 (supernatant, 1:22) was from DSHB. The antibodies to SOX1 (AF3369; 1:40), SOX2 (AF2018; 1:100) and OLIG3 (MAB32456; 1:450) were from R&D. The antibodies to NR2F1 (ABE1425; 1:500), DCX (AB2253; 1:500) and LMX1A (AB10533; 1:1,000) were from Millipore. The antibodies to SP8 (HPA054006; 1:50) and MEF2C (HPA005533; 1:100) were from Atlas Antibodies. The antibodies to CASP3 (cat. no. 9661; 1:500) and TCF7L2 (cat. no. 2569; 1:500) were from Cell Signaling. The antibody to TTR (AHP1837; 1:500) was from Bio-Rad. The antibodies to HOPX (HPA030180; 1:500), PTPRZ1 (HPA015103; 1:500) and EMX1 (HPA006421; 1:50) were from Sigma-Aldrich. Secondary Alexa Fluor antibodies 488, 546 and 647 (1:700) were obtained from Invitrogen.
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