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39037
CD44 (IM7) Rat mAb
Primary Antibodies

CD44 (IM7) Rat mAb #39037

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  1. IF

Inset A from wild-type mouse brain (left) or brain from the 5XFAD mouse model of Alzheimer's disease (right) using CD44 (IM7) Rat mAb (green) and GFAP (GA5) Mouse mAb #3670 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

Inset B from wild-type mouse brain (left) or brain from the 5XFAD mouse model of Alzheimer's disease (right) using CD44 (IM7) Rat mAb (green) and GFAP (GA5) Mouse mAb #3670 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

Confocal immunofluorescent tile scan of wild-type mouse brain (left) or brain from the 5XFAD mouse model of Alzheimer's disease (right) using CD44 (IM7) Rat mAb (green) and GFAP (GA5) Mouse mAb #3670 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue). Insets A and B are shown at higher resolution in subsequent figures.

Confocal immunofluorescent analysis of C2C12 cells (left, positive) or Neuro-2a cells (right, negative) using CD44 (IM7) Rat mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

To Purchase # 39037S
Product # Size Price
39037S
100 µl $ 268

Supporting Data

REACTIVITY M
SENSITIVITY Endogenous
MW (kDa)
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 Usage Information

Application Dilution
Immunofluorescence (Frozen) 1:50 - 1:200
Immunofluorescence (Immunocytochemistry) 1:50 - 1:200

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-Rat secondary antibodies:

  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 datasheet 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: 152

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 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 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-Rat secondary antibodies:

  6. 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 datasheet 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 November 2013

Protocol Id: 149

Specificity / Sensitivity

CD44 (IM7) Rat mAb 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:

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

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

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DyLight is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.
ProLong is a registered trademark of Life Technologies Corporation.