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95235
CD44 (IM7) Rat mAb (Alexa Fluor® 555 Conjugate)
Antibody Conjugates
Monoclonal Antibody

CD44 (IM7) Rat mAb (Alexa Fluor® 555 Conjugate) #95235

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Immunofluorescence Image 1 - CD44 (IM7) Rat mAb (Alexa Fluor® 555 Conjugate)

Confocal immunofluorescent analysis of Peyer’s patch in mouse small intestine using CD44 (IM7) Rat mAb (Alexa Fluor® 555 Conjugate) (yellow), β-Catenin (L54E2) Mouse mAb (Alexa Fluor® 488 Conjugate) #2849 (green), and CD45 (D3F8Q) Rabbit mAb (Alexa Fluor® 647 Conjugate) #81143 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

Immunofluorescence Image 1 - CD44 (IM7) Rat mAb (Alexa Fluor® 555 Conjugate)

Confocal immunofluorescent analysis of C2C12 cells (left, positive) and Neuro-2a cells (right, negative) using CD44 (IM7) Rat mAb (Alexa Fluor® 555 Conjugate) (yellow). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

Flow Cytometry Image 1 - CD44 (IM7) Rat mAb (Alexa Fluor® 555 Conjugate)

Flow cytometric analysis of LNCaP cells (blue) and HeLa cells (green) using CD44 (IM7) Rat mAb (Alexa Fluor® 555 Conjugate) (solid lines) or a concentration-matched isotype (dashed lines).

To Purchase # 95235S
Product # Size Price
95235S
100 µl  (50 tests) $ 314

Supporting Data

REACTIVITY H M
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Rat IgG2b kappa

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • 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 Description

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 555 fluorescent dye and tested in-house for direct flow cytometric analysis in human cells and immunofluorescent analysis in mouse cells and tissue. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated CD44 (IM7) Rat mAb #39037.

Product Usage Information

Application Dilution
Immunofluorescence (Frozen) 1:200
Immunofluorescence (Immunocytochemistry) 1:200
Flow Cytometry 1:50

Storage

Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

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 goat serum (#5425) / 0.3% Triton™ X-100): To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. 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. 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. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  7. 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 November 2013

Protocol Id: 222

Immunofluorescence (Immunocytochemistry)

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 goat serum (#5425) / 0.3% Triton™ X-100): To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. 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. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

  1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.

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

  2. Allow cells to fix for 15 min at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 min 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 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. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  7. 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 November 2013

Protocol Id: 182

Flow Cytometry, Live Cell Protocol for Directly Conjugated Antibodies

A. Solutions and Reagents

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

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water, mix.
  2. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.

B. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to Immunostaining.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  2. Pellet cells by centrifugation and remove supernatant.
  3. Resuspend cells in 100 µl of diluted primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
  4. Incubate for 30 min to 1 hr on ice. Protect from light.
  5. Wash by centrifugation in Antibody Dilution Buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 200-500 µl of Antibody Dilution Buffer and analyze on flow cytometer.

posted June 2017

revised June 2020

Protocol Id: 1504

Specificity / Sensitivity

CD44 (IM7) Rat mAb (Alexa Fluor® 555 Conjugate) recognizes endogenous levels of total CD44 protein. This antibody detects an epitope within the extracellular domain and is expected to detect all isoforms of CD44. In addition to mouse reactivity, this clone has shown to be positive for human samples by Flow.

Species Reactivity:

Human, Mouse

Source / Purification

This monoclonal antibody was purified from tissue culture supernatant via affinity chromatography.

Background

CD44 is a type I transmembrane glycoprotein that mediates cell-cell and cell-matrix interaction through its affinity for hyaluronic acid (HA) and possibly through other parts of the extracellular matrix (ECM). CD44 is highly polymorphic, possesses a number of alternative splice variants and undergoes extensive post-translational modifications (1,2). Increased surface levels of CD44 are characteristic of T cell activation, and expression of the protein is upregulated during the inflammatory response. Research studies have shown that interactions between CD44 and HER2 are linked to an increase in ovarian carcinoma cell growth (1-3). CD44 interacts with ezrin, radixin and moesin (ERM), linking the actin cytoskeleton to the plasma membrane and the ECM (4-6). CD44 is constitutively phosphorylated at Ser325 in resting cells. Activation of PKC results in phosphorylation of Ser291, dephosphorylation of Ser325, disassociation of ezrin from CD44, and directional motility (4).

  1. Goodison, S. et al. (1999) Mol. Pathol. 52, 189-196.
  2. Cichy, J. and Puré, E. (2003) J. Cell Biol. 161, 839-843.
  3. Bourguignon, L.Y. et al. (1997) J. Biol. Chem. 272, 27913-27918.
  4. Legg, J.W. et al. (2002) Nat. Cell Biol. 4, 399-407.
  5. Yonemura, S. et al. (1998) J. Cell Biol. 140, 885-895.
  6. Tsukita, S. et al. (1994) J. Cell Biol. 126, 391-401.

Pathways & Proteins

Explore pathways + proteins related to this product.

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

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
ProLong is a registered trademark of Life Technologies Corporation.
This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is conditioned on the buyer using the purchased product solely in research conducted by the buyer, excluding contract research or any fee for service research, and the buyer must not (1) use this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; or (c) manufacturing or quality assurance or quality control, and/or (2) sell or transfer this product or its components for resale, whether or not resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or [email protected]