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55713
CD44 (E7K2Y) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate)
Antibody Conjugates
Monoclonal Antibody
R
Recombinant

CD44 (E7K2Y) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) #55713

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  1. IHC

Immunohistochemical analysis of paraffin-embedded human serous carcinoma of the ovary using CD44 (E7K2Y) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) (red) and DAPI #4083 (blue).

Immunohistochemical analysis of paraffin-embedded human squamous cell lung carcinoma using CD44 (E7K2Y) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) (red) and DAPI #4083 (blue).
To Purchase # 55713
Cat. # Size Qty. Price
55713S
100 µl  (100 tests)

Supporting Data

REACTIVITY H M R
SENSITIVITY Endogenous
MW (kDa)
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
  • GP-Guinea Pig
  • Rab-Rabbit
  • All-All Species Expected

Product Description

This Cell Signaling Technology® antibody is conjugated to Alexa Fluor® 647 fluorescent dye under optimal conditions. This antibody conjugate is expected to exhibit the same species cross-reactivity as the unconjugated CD44 (E7K2Y) XP® Rabbit mAb #37259.

Product Usage Information

Application Dilution
Immunohistochemistry (Paraffin) 1:50 - 1:200

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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Fluorescent TUNEL Protocol for Paraffin Embedded Samples

A. Solutions and Reagents

Supplied Reagents

NOTE: Store at -20°C and avoid freeze/thaw cycles.

  1. TUNEL Equilibration Buffer (1 bottle, 5 mL)
  2. CF® Dye TUNEL Reaction Buffer (5 vials, 500 µL each): Protect from light.
  3. TdT Enzyme (1 vial, 50 µL): Supplied as 50X concentrate. Keep on ice during use.

IMPORTANT: TUNEL Equilibration Buffer and CF® Dye TUNEL Reaction Buffer contain cacodylate and cobalt chloride. Handle in accordance with the SDS and discard as toxic waste.

Additional Reagents (Not Supplied)

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

  1. Xylene
  2. Ethanol, anhydrous denatured, histological grade (100% and 95%).
  3. 10X Phosphate Buffered Saline (10X PBS): To prepare 1 L add 80 g sodium chloride (NaCl), 2 g potassium chloride (KCl), 14.4 g sodium phosphate, dibasic (Na2HPO4) and 2.4 g potassium phosphate, monobasic (KH2PO4) to 1 L dH2O. Adjust pH to 7.4.
  4. 1X Phosphate Buffered Saline (PBS): To prepare 1 L add 100 mL 10X PBS to 900 mL dH2O.
  5. 1X Phosphate Buffered Saline with Triton X-100 and BSA (PBS-TB): To prepare 100 mL 1X PBS-TB, add 10 mL 10X PBS, 200 µL Triton X-100, and 0.5 g BSA (#9998) to 90 mL dH2O, mix. Adjust pH to 7.4.
  6. Proteinase K Solution: For use dilute Proteinase K (#10012) to 20 µg/ml in dH2O.
  7. 1X Citrate Unmasking Solution (optional alternative to Proteinase K Solution): To prepare 250 ml of 1X citrate unmasking solution, dilute 25 ml of SignalStain® Citrate Unmasking Solution (10X) (#14746) with 225 ml of dH2O.

B. TUNEL Procedure

General Considerations:

  • Samples should be kept in a humidity chamber while running the TUNEL assay. Do not allow samples to dry at any time during this procedure.
  • All subsequent incubations should be carried out at room temperature (20-25°C) unless noted otherwise.
  • Heat-mediated antigen retrieval is recommended if co-labeling with an antibody is attempted.

Sample Preparation

  1. Deparaffinize/rehydrate:
    1. Incubate sections in three washes of xylene for 5 min each.
    2. Incubate sections in two washes of 100% ethanol for 10 min each.
    3. Incubate sections in two washes of 95% ethanol for 10 min each.
  2. Wash: Rinse two times in PBS for 5 min each.
  3. Antigen Retrieval: Incubate samples with Proteinase K solution for 30 min. Incubation time and temperature may require optimization depending on tissue type.
    1. Alternatively Citrate Unmasking Solution can be used: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at sub-boiling temperature (95°-98°). Cool slides on bench top for 30 min.
  4. Wash: Rinse two times in PBS for 5 min each.

TUNEL Reaction

  1. Preparation: Incubate samples with 100 µL TUNEL Equilibration Buffer or enough to cover the sample for 5 min.
  2. Detection: Immediately before use, prepare TUNEL reaction mix by adding 1 µL of TdT Enzyme to 50 µL of TUNEL Reaction Buffer for each labeling reaction. Remove Equilibration Buffer and add 50 µL (or enough to cover the sample) of TUNEL reaction mix to each sample.
  3. Incubate: Incubate for 60 min at 37°C, protected from light. Tissue sections may require 2 hours of incubation at 37°C also protected from light.
  4. Wash: Rinse three times in PBS-TB for 5 min each.
  5. Co-label: If desired, proceed with immunostaining for co-labeling. Consult with the manufacturer's protocol for the recommended staining conditions.
  6. Mount: Mount samples in a compatible anti-fade medium such ProLong® Gold Antifade Reagent (#9071).

Protocol Id: 2624

Specificity / Sensitivity

CD44 (E7K2Y) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) recognizes endogenous levels of total CD44 protein.

Species Reactivity:

Human, Mouse, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro136 of human CD44 protein. This sequence region is conserved in all isoforms of CD44 reported in Uniprot, with the exception of isoform 2 and isoform 19.

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).

Limited Uses

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Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not for Use in Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
XP is a registered trademark of Cell Signaling Technology, Inc.
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].
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