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50490
CTLA-4 (E5S7E) Rabbit mAb
Primary Antibodies
Monoclonal Antibody

CTLA-4 (E5S7E) Rabbit mAb #50490

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  1. IF
Immunofluorescence Image 1: CTLA-4 (E5S7E) Rabbit mAb
Confocal immunofluorescent analysis of fixed frozen mouse spleen, labeled with CTLA-4 (E5S7E) Rabbit mAb (left, red) and co-labeled with CD4 (RM4-5) Rat mAb (FITC Conjugate) #96127 (right, green) and DAPI #4083 (right, blue).
Immunofluorescence Image 2: CTLA-4 (E5S7E) Rabbit mAb
Confocal immunofluorescent analysis of fixed frozen mouse thymus, labeled with CTLA-4 (E5S7E) Rabbit mAb (left, red) and co-labeled with CD4 (RM4-5) Rat mAb (FITC Conjugate) #96127 (right, green) and DAPI #4083 (right, blue).
Immunofluorescence Image 3: CTLA-4 (E5S7E) Rabbit mAb
Confocal immunofluorescent analysis of Peyer's patch in fixed frozen mouse colon, labeled with CTLA-4 (E5S7E) Rabbit mAb (left, red) and co-labeled with CD4 (RM4-5) Rat mAb (FITC Conjugate) #96127 (right, green) and DAPI #4083 (right, blue).
To Purchase # 50490
Cat. # Size Qty. Price
50490S
100 µl
$ 287

Supporting Data

REACTIVITY M
SENSITIVITY Endogenous
MW (kDa) 30-40
Source/Isotype Rabbit IgG

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • C&R-CUT&RUN
  • C&T-CUT&Tag
  • DB-Dot Blot
  • eCLIP-eCLIP
  • IF-Immunofluorescence
  • F-Flow Cytometry

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Vir-Virus
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Usage Information

Application Dilution
Immunofluorescence (Frozen) 1:200 - 1:800

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/mL BSA, 50% glycerol, and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Protocol

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Immunofluorescence (Frozen)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. Blocking Buffer: (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425) to 9.5 ml 1X PBS) and mix well. While stirring, add 30 µl Triton™ X-100.
  4. Antibody Dilution Buffer: (1X PBS / 1% BSA / 0.3% Triton™ X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (#9998), mix.
  5. Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:

    NOTE: When using any primary or fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to determine which dilution allows for the strongest specific signal with the least background for your sample.

  6. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Frozen/Cryostat Sections (IF-F)

  1. For fixed frozen tissue proceed with Immunostaining (Section C).
  2. For fresh, unfixed frozen tissue, please fix immediately, as follows:
    1. Cover sections with 4% formaldehyde dilute in 1X PBS.

      NOTE: Formaldehyde is toxic, use only in fume hood.

    2. Allow sections to fix for 15 minutes at room temperature.
    3. Aspirate liquid, rinse three times in 1X PBS for 5 minutes each.
    4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Block specimen in Blocking Buffer for 60 min.
  2. While blocking, prepare primary antibody by diluting as indicated on protocol on product webpage in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.
  6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hr at room temperature in the dark.
  7. Rinse three times in 1X PBS for 5 min each.
  8. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

posted November 2006

revised July 2016

Protocol Id: 151

Specificity / Sensitivity

CTLA-4 (E5S7E) Rabbit mAb recognizes endogenous levels of total mouse CTLA-4 protein.

Species Reactivity:

Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the extracellular domain of mouse CTLA-4 protein.

Background

Cytotoxic T-lymphocyte protein 4 (CTLA-4, CD152) is an Ig superfamily member that negatively regulates early T cell activation (1-4). The CTLA-4 protein is primarily expressed on T cells, including CD8+ cytotoxic T cells, CD4+ helper T cells, and CD4+/FoxP3+ regulatory T cells (1,2). CTLA-4 protein competes with CD28 for B7.1 (CD80) and B7.2 (CD86) binding at the cell surface, which results in the downregulation of T cell activity (5). The activation of SHP-2 and PP2A downstream of CTLA-4 attenuates TCR signaling (6). Research studies indicate that CTLA4 knockout mice display lymphoproliferative disorders leading to early death, confirming the role of CTLA-4 as a negative regulator of T cells (7). Mutations in the corresponding CTLA4 gene are associated with multiple disorders, including insulin-dependent diabetes mellitus, Graves' disease, Hashimoto thyroiditis, celiac disease, systemic lupus erythematosus, and type V autoimmune lymphoproliferative syndrome (8,9). Additional studies demonstrate that CTLA-4 blockade is an effective strategy for tumor immunotherapy (10-12).
  1. Brunet, J.F. et al. (1987) Nature 328, 267-70.
  2. Brunet, J.F. et al. (1988) Immunol Rev 103, 21-36.
  3. Dariavach, P. et al. (1988) Eur J Immunol 18, 1901-5.
  4. Linsley, P.S. (1995) J Exp Med 182, 289-92.
  5. Collins, A.V. et al. (2002) Immunity 17, 201-10.
  6. Rudd, C.E. et al. (2009) Immunol Rev 229, 12-26.
  7. Waterhouse, P. et al. (1995) Science 270, 985-8.
  8. Romo-Tena, J. et al. (2013) Autoimmun Rev 12, 1171-6.
  9. Wang, J. et al. (2014) PLoS One 9, e85982.
  10. Egen, J.G. et al. (2002) Nat Immunol 3, 611-8.
  11. Hodi, F.S. et al. (2003) Proc Natl Acad Sci U S A 100, 4712-7.
  12. Pardoll, D.M. (2012) Nat Rev Cancer 12, 252-64.

Pathways & Proteins

Explore pathways + proteins related to this product.

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For Research Use Only. Not For Use In Diagnostic Procedures.
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