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Ab37305

Manufactured by Abcam
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Sourced in Japan, United Kingdom

Ab37305 is a monoclonal antibody that recognizes the Pax3 protein. Pax3 is a transcription factor involved in regulating embryonic development and stem cell differentiation. The antibody is suitable for use in various applications, including immunohistochemistry, immunocytochemistry, and Western blotting.

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Lab products found in correlation

Erpr7511, by Abcam (4 mentions) Cat5 h10, by Zymo Research (3 mentions) A5441, by Merck Group (2 mentions) Sc 13515, by Santa Cruz Biotechnology (2 mentions) Sc 13596, by Santa Cruz Biotechnology (2 mentions) A11035, by Thermo Fisher Scientific (2 mentions) Eclipse e800, by Nikon (2 mentions) A11034, by Thermo Fisher Scientific (2 mentions) A0398, by Agilent Technologies (2 mentions) P8920, by Merck Group (2 mentions) Caspase 3, by Cell Signaling Technology (2 mentions) M71165, by Agilent Technologies (2 mentions) Mem m6 1, by LifeSpan BioSciences (2 mentions) Biotinylated antibodycoupled to streptavidin peroxidase complex, by Vector Laboratories (2 mentions) Streptavidin peroxidase, by Vector Laboratories (2 mentions) Human cd44h 2c5, by R&D Systems (2 mentions) M7165, by Agilent Technologies (2 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Permount, by Thermo Fisher Scientific (1 mentions) Normal mouse igg, by Agilent Technologies (1 mentions)

13 protocols using ab37305

1

Immunostaining Protocol for Bcl9 and β-Catenin

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3-Aminopropyltriethoxysilane (APS), bovine serum albumin (BSA, essentially fatty acid- and globulin-free), 30% Brij® L23 solution, normal goat IgG and rabbit IgG were purchased from Sigma-Aldrich (St. Louis, MO, USA). Normal mouse IgG was from Dako (Glostrup, Denmark). Mouse monoclonal anti-human Bcl9 (BMR00368; 2.0 μg/ml) was a gift from Bio Matrix Research Inc. (Nagareyama, Chiba, Japan) and rabbit polyclonal anti-human Bcl9 (ab37305; 5.0 μg/ml) was purchased from Abcam (Cambridge, MA, USA) [12 (link), 30 ]. Mouse monoclonal anti-β-catenin (CTNNB1, UM500015; 1.33 μg/ml) was purchased from OriGene (Rockville, MD, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (AP308P; 10.0 μg/ml) and HRP-conjugated goat anti-rabbit IgG (AP307P; 10.0 μg/ml) antibodies were from Millipore (Temecula, CA, USA). 3,3'-Diaminobenzidine-4HCl (DAB) was from Dojin Chemical Co. (Kumamoto, Japan). Permount was purchased from Thermo Fisher Scientific (Hudson, NH, USA). All other reagents used in this study were from Wako Pure Chemicals (Osaka, Japan).
Soe M.T., Shibata Y., Win Htun M., Abe K., Soe K., Win Than N., Lwin T., Phone Kyaw M, & Koji T. (2019). Immunohistochemical Mapping of Bcl9 Using Two Antibodies that Recognize Different Epitopes Is Useful to Characterize Juvenile Development of Hepatocellular Carcinoma in Myanmar. Acta Histochemica et Cytochemica, 52(1), 9-17.
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2

Immunohistochemical Analysis of BCL9 and Hypoxia Markers

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Immunnohistochemical staining for BCL9 was performed by using the standard histological procedure described in the manual for Histostain-Plus IHC Kit, DAB (Phygene). BCL9 staining was scored according to two investigators with previous protocols as negative (−), weak positive (+), and strong (++). The results were analyzed by standard light microscopy. Scoring was assessed blindly with respect to the histologic grade of HCC specimens. Immunofluorescence staining was performed with primary and secondary antibodies diluted in 10% BSA, and the nucleus was stained by DAPI (Sigma). The antibody against HIF-1α (sc-13515, Santa Cruz Biotechnology, 1:500), antibody against HIF-2α (sc-13596, Santa Cruz Biotechnology, 1:500), antibody against BCL9 (ab37305, Abcam, 1:500 dilution) and antibody against β-actin (A5441, Sigma) were used in this study. The antibodies are anti-rabbit in this study. All fluorescent secondary antibodies were used at a dilution of 1:200 for 30 min (invitrogen). Quantification of immunofluorescence was performed using NIH Image J.
Xu W., Zhou W., Cheng M., Wang J., Liu Z., He S., Luo X., Huang W., Chen T., Yan W, & Xiao J. (2017). Hypoxia activates Wnt/β-catenin signaling by regulating the expression of BCL9 in human hepatocellular carcinoma. Scientific Reports, 7, 40446.
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3

Immunofluorescence Staining of Cells

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Single-cell suspensions were spun onto glass slides using a cytocentrifuge
(Shandon) or were grown on polylysine-coated slides (p8920, Sigma), as described13 (link). Cells were fixed at room temperature in 2%
paraformaldehyde for 20 min, permeabilized in TBS-Tween 20 for 20 min, washed three times
in PBS, and then blocked with 5% bovine serum albumin in PBS for 2 h before addition of
primary antibodies against BCL9 (ab37305, Abcam, 1:200), β-catenin (CAT5-H10,
Zymed, 1:100), Myeloperoxidase (A0398, DAKO, 1:100), CyPA (ERPR7511, Abcam, 1:200), Alexa
Fluor 647-conjugated CD147 (HIM6, Biolegend, 1:200), or FITC-conjugated CD138 (MI15,
Becton Dickinson, 1:200). Cells were incubated overnight with primary antibodies at 4
°C, and then washed three times in PBS before staining with secondary antibodies
conjugated to Alexa Fluor 488 (A11034, Molecular Probes, 1:200) or Alexa Fluor 546
(A11035, Molecular Probes, 1:200). Images were acquired with the aid of a Bio-Rad Radiance
2000 laser scanning confocal or Nikon Eclipse E800 phase-contrast microscope. Optimal
antibody concentrations were used according to manufacturer's recomendations.
Zhu D., Wang Z., Zhao J.J., Calimeri T., Meng J., Hideshima T., Fulciniti M., Kang Y., Ficarro S., Tai Y.T., Hunter Z., McMilin D., Tong H., Mitsiades C.S., Wu C., Treon S., Dorfman D.M., Pinkus G., Munshi N., Tassone P., Marto J., Anderson K, & Carrasco R.D. (2015). The Cyclophilin A-CD147 complex promotes bone marrow colonization of B-cell malignancies: implications for therapy. Nature medicine, 21(6), 572-580.
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4

Western Blot Analysis of Apoptosis Markers

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Total proteins from tissues and cells were lysed conforming to the user’s guidebook of RIPA lysis buffer (Beyotime, China); this was followed by separation with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transfer with polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). After that, membranes were subjected to a standard blocking with 5% non-fat milk, hybridization with primary antibodies at 4 °C overnight, and incubation with secondary antibodies at room temperature for one hour. The bands were detected according to the instructions of the ECL detection kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The primary antibodies are presented as follows: Bcl-2 (Bcl-2, 1:1000; ab32124, Abcam, Cambridge, UK), Bcl-9 (Bcl-9, 1:1000; ab37305, Abcam, Cambridge, UK), caspase-3 (caspase-3, 1:1000; ab13847, Abcam), caspase-7 (caspase-7, 1:1,000; ab255818, Abcam, Cambridge, UK), cleaved caspase-3 (cleaved caspase-3, 1:1000; #9661, Cell Signaling Technology, Boston, MA, USA), cleaved caspase-7 (cleaved caspase-7, 1:800; #8438, Cell Signaling Technology, Boston, MA, USA), bax (bax, 1:1000; ab32503, Abcam, Cambridge, UK), and goat anti-rabbit IgG (H + L) secondary antibody (1:5000; ab6721, Abcam, Cambridge, UK ).
Wang Y., Guo Y., Duan C., Yang R., Zhang L., Liu Y, & Zhang Y. (2022). Long Non-Coding RNA GDAR Regulates Ovine Granulosa Cells Apoptosis by Affecting the Expression of Apoptosis-Related Genes. International Journal of Molecular Sciences, 23(9), 5183.
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5

Immunohistochemical Analysis of Human Tissues

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Tissue sections were processed as described13 (link). Human tissue samples were obtained from the Department of Pathology,
Brigham and Women's Hospital. Sections were incubated with primary antibodies (5 μg
per ml) or the corresponding IgG fraction of pre-immune serum overnight at 4 °C in
blocking solution (3% BSA in PBS). Anti-human primary specific antibodies included: BCL9
(ab37305, Abcam, 1:200), CD138 (PN IM 2757, Beckman Coulter, 1:1000), CD34 (M71165, DAKO,
1:400), ERG (5115-1, Epitomics, 1:3000), Caspase-3 (9664, Cell Signaling, 1:150), CD147
(MEM-M6-1, LifeSpan BioSciences, 1:300), CyPA (ERPR7511, Abcam, 1:1500) and CyPB (AV44365,
Sigma, 1:100); and were visualized with the aid of the corresponding biotinylated antibody
coupled to streptavidin-peroxidase complex (Vector Labs). For CD147 immunohistochemistry,
non-decalcified BM clots were used. For negative controls, tissue sections were incubated
in the absence of primary antibodies or pre-immune serum from the species of origin of the
primary antibody. Optimal antibody concentrations were used according to manufacturer
recomendations.
Zhu D., Wang Z., Zhao J.J., Calimeri T., Meng J., Hideshima T., Fulciniti M., Kang Y., Ficarro S., Tai Y.T., Hunter Z., McMilin D., Tong H., Mitsiades C.S., Wu C., Treon S., Dorfman D.M., Pinkus G., Munshi N., Tassone P., Marto J., Anderson K, & Carrasco R.D. (2015). The Cyclophilin A-CD147 complex promotes bone marrow colonization of B-cell malignancies: implications for therapy. Nature medicine, 21(6), 572-580.
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6

Quantitative Analysis of BCL9 Expression

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MiRs quantitative reverse transcriptase-PCR (Q-RT-PCR) was performed according to manufacturer’s instruction (Applied Biosystem). U44 primer from ABI was used as an internal control. Q-RT-PCR was performed for evaluation of BCL9 mRNA levels as previously described (9 (link), 20 (link)), and GAPDH severed as an internal control. The primers of BCL9, Axin 2, and CD44 are listed in Table S1. Western blot, miRs-LNA ISH, immunofluorescence (IF), and immunohistochemistry (IHC) were carried out as previously described (21 (link)). Antibodies included: BCL9 (6109) antibody for western blotting and IF (10 ); Alexa Fluor 546 goat anti-rabbit IgG (A-11010, Invitrogen) as secondary antibody for IF; as well as actin-HRP (C-11, Santa Cruz) and anti-Rabbit IgG HRP conjugated secondary antibodies (W401b, Promega) for western blotting; as well as BCL9 (ab37305, Abcam), Caspase-3 (#9662, Cell Signaling), Ki-67 (NB110-90592, Novus), Axin-2 (#2151, Cell Signaling), CD44 (#5640s, Cell Signaling), and BCL6 (ab9479, Abcam) were used for IHC.
Zhao J.J., Lin J., Zhu D., Wang X., Brooks D., Chen M., Chu Z.B., Takada K., Ciccarelli B., Admin S., Tao J., Tai Y.T., Treon S., Pinkus G., Kuo W.P., Hideshima T., Bouxsein M., Munshi N., Anderson K, & Carrasco R. (2014). miR-30-5p functions as a tumor suppressor and novel therapeutic tool by targeting the oncogenic Wnt/β-catenin/BCL9 pathway. Cancer research, 74(6), 1801-1813.
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7

Protein Level Analysis via Western Blot

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Standard Western-blot assays were used to analyze the levels of protein. Antibodies against HIF-1α (sc-13515, Santa Cruz Biotechnology, 1:500), anti-HIF-2α (sc-13596, Santa Cruz Biotechnology, 1:500), anti-BCL-9 (ab37305, Abcam, 1:500 dilution) and anti–β-actin (A5441, Sigma) antibodies were used in this study.
Tan Z., Huang Q., Zang J., Teng S.F., Chen T.R., Wei H.F., Song D.W., Liu T.L., Yang X.H., Fu C.G., Hu Z.Q., Zhou W., Yan W.J, & Xiao J.R. (2016). HIF-1α activates hypoxia-induced BCL-9 expression in human colorectal cancer cells. Oncotarget, 8(16), 25885-25896.
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8

Quantifying Angiogenesis in Cancer Models

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Example 7

Tissue sections were process as described (Sukhdeo, K. et al. Proc Natl Acad Sci USA 104, 7516-21 (2007)). Sections were incubated with primary antibodies (5 μg/ml) or the corresponding IgG fraction of preimmune serum overnight at 4° C. in blocking solution (3% BSA/PBS). BCL9 (ab37305, Abcam), mouse CD34 (RAM34, eBiosciences), human CD34 (M7165, Dako) and human CD44H (2C5, R&D Systems) antibodies were employed. Blood vessel formation in the CRC and MM models was evaluated using anti-mouse CD34 and anti-human CD34 antibodies, respectively, and the corresponding biotinylated antibodies coupled to streptavidin peroxidase (Vector). The number of blood vessels was determined by counting the mean number of independent blood vessels in 5 randomly selected fields at 50× magnification as highlighted by CD34 staining (brown color). See results in FIGS. 4b, 4e, 4i, 6.

US10703785B2. Targeting deregulated Wnt signaling in cancer using stabilized alpha-helices of BCL-9 (2020-07-07). None [US], None [US], None [US]. Inventors: Loren D. Walensky [US], Ruben Carrasco [US], Gregory H. Bird [US].
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9

Immunohistochemical Analysis of Blood Vessels

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Example 7

Tissue sections were process as described (Sukhdeo, K. et al. Proc Natl Acad Sci USA 104, 7516-21 (2007)). Sections were incubated with primary antibodies (5 μg/ml) or the corresponding IgG fraction of preimmune serum overnight at 4° C. in blocking solution (3% BSA/PBS). BCL9 (ab37305, Abcam), mouse CD34 (RAM34, eBiosciences), human CD34 (M7165, Dako) and human CD44H (2C5, R&D Systems) antibodies were employed. Blood vessel formation in the CRC and MM models was evaluated using anti-mouse CD34 and anti-human CD34 antibodies, respectively, and the corresponding biotinylated antibodies coupled to streptavidin peroxidase (Vector). The number of blood vessels was determined by counting the mean number of independent blood vessels in 5 randomly selected fields at 50× magnification as highlighted by CD34 staining (brown color). See results in FIG. 4B, FIG. 4E, FIG. 4I, FIG. 6.

US11220532B2. Targeting deregulated Wnt signaling in cancer using stabilized alpha-helices of BCL-9 (2022-01-11). DANA-FARBER CANCER INSTITUTE, INC. [US]. Inventors: Loren D. Walensky [US], Ruben Carrasco [US], Gregory H. Bird [US].
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10

Immunofluorescence Staining of Cells

Cited 1 time
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Single-cell suspensions were spun onto glass slides using a cytocentrifuge
(Shandon) or were grown on polylysine-coated slides (p8920, Sigma), as described13 (link). Cells were fixed at room temperature in 2%
paraformaldehyde for 20 min, permeabilized in TBS-Tween 20 for 20 min, washed three times
in PBS, and then blocked with 5% bovine serum albumin in PBS for 2 h before addition of
primary antibodies against BCL9 (ab37305, Abcam, 1:200), β-catenin (CAT5-H10,
Zymed, 1:100), Myeloperoxidase (A0398, DAKO, 1:100), CyPA (ERPR7511, Abcam, 1:200), Alexa
Fluor 647-conjugated CD147 (HIM6, Biolegend, 1:200), or FITC-conjugated CD138 (MI15,
Becton Dickinson, 1:200). Cells were incubated overnight with primary antibodies at 4
°C, and then washed three times in PBS before staining with secondary antibodies
conjugated to Alexa Fluor 488 (A11034, Molecular Probes, 1:200) or Alexa Fluor 546
(A11035, Molecular Probes, 1:200). Images were acquired with the aid of a Bio-Rad Radiance
2000 laser scanning confocal or Nikon Eclipse E800 phase-contrast microscope. Optimal
antibody concentrations were used according to manufacturer's recomendations.
Zhu D., Wang Z., Zhao J.J., Calimeri T., Meng J., Hideshima T., Fulciniti M., Kang Y., Ficarro S., Tai Y.T., Hunter Z., McMilin D., Tong H., Mitsiades C.S., Wu C., Treon S., Dorfman D.M., Pinkus G., Munshi N., Tassone P., Marto J., Anderson K, & Carrasco R.D. (2015). The Cyclophilin A-CD147 complex promotes bone marrow colonization of B-cell malignancies: implications for therapy. Nature medicine, 21(6), 572-580.
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