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71536
Keratin 5 (E2T4B) XP® Rabbit mAb
Primary Antibodies
Monoclonal Antibody
R
Recombinant

Keratin 5 (E2T4B) XP® Rabbit mAb #71536

Citations (8)
Filter:
  1. IHC
  2. IF
Immunohistochemical analysis of paraffin-embedded human mucoepidermoid carcinoma of the larynx using Keratin 5 (E2T4B) XP® Rabbit mAb performed on the Leica® BOND Rx.
Immunohistochemical analysis of paraffin-embedded human squamous carcinoma of the tongue using Keratin 5 (E2T4B) XP® Rabbit mAb performed on the Leica® BOND Rx.
Immunohistochemical analysis of paraffin-embedded human endometrioid adenocarcinoma using Keratin 5 (E2T4B) XP® Rabbit mAb performed on the Leica® BOND Rx.
Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using Keratin 5 (E2T4B) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human ovarian serous carcinoma using Keratin 5 (E2T4B) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded normal mouse tissues: skin (top-left), lung (top-right), colon (bottom-left), and pancreas (bottom-right) using Keratin 5 (E2T4B) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human squamous cell lung carcinoma using Keratin 5 (E2T4B) XP® Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).
Immunohistochemical analysis of paraffin-embedded A-431 cell pellet (left, positive) or SNB19 cell pellet (right, negative) using Keratin 5 (E2T4B) XP® Rabbit mAb.
Confocal immunofluorescent analysis of fixed frozen mouse skin labeled with Keratin 5 (E2T4B) XP® Rabbit mAb (left, red), autofluorescence (right, green), and DAPI #4083 (right, blue).
Confocal immunofluorescent analysis of BxPC-3 cells (left, positive) and PANC-1 cells (right, negative) using Keratin 5 (E2T4B) XP® Rabbit mAb (green) and DAPI #4083 (blue).
To Purchase # 71536
Cat. # Size Qty. Price
71536T
20 µl
71536S
100 µl

Supporting Data

REACTIVITY H M
SENSITIVITY Endogenous
MW (kDa) 62
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 Usage Information

Application Dilution
IHC Leica Bond 1:125 - 1:500
Immunohistochemistry (Paraffin) 1:250 - 1:1000
Immunofluorescence (Frozen) 1:100 - 1:400
Immunofluorescence (Immunocytochemistry) 1:400 - 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.

For a carrier-free (BSA and azide free) version of this product see product #81817.

Protocol

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Immunohistochemistry (Leica® BOND™)

NOTE: Please see product datasheet or product webpage for appropriate antibody dilution^.

  Step Reagents Time/Temperature
1 Dewax BOND™ Dewax Solution, 100% Alcohol, BOND™ Wash Solution Pre-programmed Leica® BOND™ 
2 Antigen Retrieval  BOND™ Epitope Retrieval ER2 Solution 20 min., 100˚C | Protocol: HIER 20 min with ER2
3 Peroxide Block Refine Detection Kit Peroxide Block* 5 min.
  WASH BOND™ Wash Solution  3x 0:00 min.
4 Protein Block (optional) #5425 NGS or #15019 Animal-Free Blocking Solution   20 min. 
5 Primary Antibody^ Dilute in #8112 SignalStain® Antibody Diluent 30 min. 
  WASH BOND™ Wash Solution  3x 2:00 min.
NA Post Primary Mouse Linker Refine Detection Kit Post Primary*  Not Applied 
6 Secondary Detection Refine Detection Kit Polymer*  10 min. 
  WASH BOND™ Wash Solution/Deionized Water Custom (see below)
7a Visualization Refine Detection Kit Mixed DAB Refine*  0:00 min. 
7b Visualization Refine Detection Kit Mixed DAB Refine*  10 min. 
  WASH Deionized Water 3x 0:00 min.
8 Counterstain Refine Detection Kit Hematoxylin*  5 min. 
  WASH Deionized Water 0:00 min. 
  WASH BOND™ Wash Solution  0:00 min. 
  WASH Deionized Water 0:00 min. 
9 Dehydration (Offline):    
  Incubate sections in 95% ethanol two times for 10 seconds each.  
  Repeat in 100% ethanol, incubating sections two times for 10 seconds each.  
  Repeat in xylene, incubating sections two times for 10 seconds each.  
10 Mount sections with coverslips and #14177 SignalStain® Mounting Medium  
       
  Optional Custom wash:  BOND™ Wash Solution 2:00
    BOND™ Wash Solution Dispenser Type: OPEN 0:00
    BOND™ Wash Solution 2:00
    BOND™ Wash Solution Dispenser Type: OPEN 0:00
    BOND™ Wash Solution 0:00
    Deionized Water 0:00

*Reagent included in BOND™ Polymer Refine Detection Kit (Catalog No: DS9800)

LEICA® is a registered trademark of Leica Microsystems IR GmbH.

BOND™ is a trademark of Leica Biosystems Melbourne Pty. Ltd. No affiliation or sponsorship between CST and Leica Microsystems IR GmbH or Leica Biosystems Melbourne Pty. Ltd is implied.

posted August 2018

revised September 2018

Protocol Id: 1444

Immunohistochemistry (Paraffin)

A. Solutions and Reagents

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

  1. Xylene.
  2. Ethanol, anhydrous denatured, histological grade (100% and 95%).
  3. Deionized water (dH2O).
  4. Hematoxylin (optional).
  5. Wash Buffer:
    1. 1X Tris Buffered Saline with Tween® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween® 20 (#9997) to 900 ml dH20, mix.
  6. SignalStain® Antibody Diluent (#8112).
  7. 1X Citrate Unmasking 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.
  8. 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H2O2 to 90 ml dH2O.
  9. Blocking Solution: TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
    1. TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
    2. 1X Animal-Free Blocking Solution: to 4 mL of dH2O add 1 ml of Animal-Free Blocking Solution (5X) (#15019).
  10. Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Rabbit #8114).
  11. Substrate: SignalStain® DAB Substrate Kit (#8059).
  12. Hematoxylin: Hematoxylin (#14166).
  13. Mounting Medium: SignalStain® Mounting Medium (#14177).

B. Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

  1. Deparaffinize/hydrate sections:
    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 sections two times in dH2O for 5 min each.

C. Antigen Unmasking

For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

D. Staining

  1. Wash sections in dH2O three times for 5 min each.
  2. Incubate sections in 3% hydrogen peroxide for 10 min.
  3. Wash sections in dH2O two times for 5 min each.
  4. Wash sections in wash buffer for 5 min.
  5. Block each section with 100–400 µl of preferred blocking solution for 1 hr at room temperature.
  6. Remove blocking solution and add 100–400 µl primary antibody diluted in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
  7. Equilibrate SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) to room temperature.
  8. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
  9. Cover section with 1–3 drops SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) as needed. Incubate in a humidified chamber for 30 min at room temperature.
  10. Wash sections three times with wash buffer for 5 min each.
  11. Add 1 drop (30 µl) SignalStain® DAB Chromogen Concentrate to 1 ml SignalStain® DAB Diluent and mix well before use.
  12. Apply 100–400 µl SignalStain® DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
  13. Immerse slides in dH2O.
  14. If desired, counterstain sections with hematoxylin (#14166).
  15. Wash sections in dH2O two times for 5 min each.
  16. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 sec each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 sec each.
    3. Repeat in xylene, incubating sections two times for 10 sec each.
  17. Mount sections with coverslips and mounting medium (#14177).

DETECTION REAGENT/SUBSTRATE COMPATIBILITY
RECOMMENDED
DETECTION REAGENTS
SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) #8114 SignalStain® Boost IHC Detection Reagent (AP, Rabbit) #18653
COMPATIBLE
CHROMOGEN
SignalStain® DAB Substrate Kit #8059 SignalStain® Vibrant Red Alkaline Phosphatase Substrate Kit #76713
SignalStain® Vivid Purple Peroxidase Substrate Kit #96632 SignalStain® Ultra Blue Alkaline Phosphatase Substrate Kit #12824
SignalStain® Deep Black Peroxidase Substrate Kit #72986  
SignalStain® Radiant Yellow Peroxidase Substrate Kit #69644  

NOTE: Use of detection reagents other than those specified in this protocol may require further optimization of the primary antibody to account for the different sensitivities of the detection reagents.


posted February 2010

revised June 2020

Protocol Id: 283

Immunofluorescence Protocol for Frozen Tissue (BSA-free)

IMPORTANT: This protocol employs a strategy with which Bovine Serum Albumin (BSA) is removed from the Antibody Dilution Buffer. Validation testing has revealed that this strategy reduces performance issues caused by interaction with some lots of BSA. Where appropriate, this protocol will be linked to its validated antibody under the Product Information banner on the product-specific webpage.

A. Solutions and Reagents

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

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1L 1X PBS, add 100 mL 10X Wash Buffer, Phosphate Buffered Saline (#12528) to 900 mL dH2O, mix. Adjust pH to 8.0.
  2. 4% Formaldehyde, Methanol-Free (#47746): Use fresh.
  3. Blocking Buffer: Purchase ready-to-use Immunofluorescence Blocking Buffer (#12411), or prepare a 1X PBS / 5% normal serum / 0.3% Triton X-100 buffer by adding 0.5 mL normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 30 µL Triton X-100 to 9.5 mL 1X PBS. Store at 4°C.
  4. BSA-Free Antibody Dilution Buffer : Prepare a 1X PBS / 0.3% Triton X-100 buffer by adding 30 µL Triton X-100 to 10 mL 1X PBS. Store at 4°C.
  5. Fluorochrome-conjugated Secondary Antibody: Use a secondary antibody that is reactive to your primary antibody host species (e.g., rabbit). Click here for a list of secondary antibodies approved for immunofluorescence.

B. Fixation

  1. For fixed frozen tissue proceed with Immunostaining (Section C).
  2. For fresh, unfixed frozen tissue, fix immediately, as follows:
    1. Cover sections to a depth of 2–3 mm with 4% formaldehyde.
    2. Allow sections to fix for 15 min at room temperature.
    3. Rinse slides three times in PBS for 5 min each.
    4. Proceed with Immunostaining (Section C).

C. Immunostaining

  1. Block specimen in Blocking Buffer for 60 min.
  2. While blocking, dilute primary antibody in BSA-Free Antibody Dilution Buffer (see product website for recommended dilution range).
  3. Aspirate blocking solution then apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.
  6. NOTE: If using a fluorochrome-conjugated primary antibody, then skip to Section C, Step 8.

  7. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in BSA-Free Antibody Dilution Buffer for 1–2 hours protected from light.
  8. Rinse three times in 1X PBS for 5 min each protected from light.
  9. Counterstain as appropriate.
  10. NOTE: When including fluorescent cellular dyes in your experiment (DNA dyes, etc.), please refer to the dye product page for its recommended protocol. View our listing of cellular dyes validated for use in immunofluorescence.

  11. Mount samples for imaging.
  12. For long-term storage, store samples at 4°C protected from light.

Protocol Id: 2485

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)) and 0.5 mL 20X PBS to 9.0 mL dH2O, mix well. While stirring, add 30 µl Triton X-100.
  4. Antibody Dilution Buffer (1X PBS / 0.3% Triton X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well.
  5. Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 555 Conjugate) #4413
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) #8889
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414
  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 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 September 2016

Protocol Id: 1204

Specificity / Sensitivity

Keratin 5 (E2T4B) XP® Rabbit mAb recognizes endogenous levels of total keratin 5 protein.

Species Reactivity:

Human, Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val578 of human keratin 5 protein.

Background

Keratins (cytokeratins) are intermediate filament proteins that are mainly expressed in epithelial cells. Keratin heterodimers composed of an acidic keratin (or type I keratin, keratins K9-K28) and a basic keratin (or type II keratin, keratins K1-K8 and K71-K80) assemble to form filaments. Keratin isoforms demonstrate tissue- and differentiation-specific profiles that make them useful as research and clinical biomarkers (1,2).

Dysregulation/mutations in keratin genes can lead to a variety of disorders affecting the skin, hair, nails, and other epithelial tissues (3). While expression of keratins can be variable, immunohistochemical staining of keratins is widely used to help in the identification and classification of epithelial tumors, and may also provide prognostic information.

Keratins 8 and 18 (K8/K18) are expressed in simple epithelia of normal tissue, as well as in adenocarcinomas of the breast, lung, ovary, and gastrointestinal tract. Keratin 17 is expressed in basal keratinocytes of stratified epithelia, hair follicles, and sebaceous glands. Onset of keratin 17 expression coincides with the definition of major epithelial lineages during skin development (4). Keratin 14 (K14) is expressed in basal cells of stratified epithelia, and in basal-like subtypes of breast cancer and squamous cell carcinomas. Keratin 19 (K19) is expressed in glandular epithelia, including the liver, gallbladder, and pancreas, as well as in adenocarcinomas of the breast, thyroid, and bile duct. Keratin 20 (K20) is expressed in gastrointestinal epithelium, urothelium, and Merkel cells in the skin, as well as in colorectal carcinomas and some urothelial carcinomas. Keratin 5/6 (K5/6) is expressed in basal cells of stratified epithelia, including the skin, prostate, and breast, as well as in basal-like breast cancers, squamous cell carcinomas, and some lung carcinomas. Keratin 7 (K7) is expressed in glandular epithelia, such as those in the lung, breast, and female reproductive tract, as well as in adenocarcinomas of the lung, breast, and ovary (5,6).

Keratins, particularly K8, K18, and K19, serve as biomarkers for identification of circulating tumor cells (CTCs) (5).

Post-translational modifications, including phosphorylation, acetylation, ubiquitylation, sumoylation, glycosylation, and transamidation, have been shown to affect the functions of keratins in normal and disease states (6). Understanding the molecular mechanisms underlying these PTMs may provide insights into cancer pathogenesis.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

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.
SignalStain is a registered trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
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