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61193
MUC5AC (E3O9I) XP® Rabbit mAb

MUC5AC (E3O9I) XP® Rabbit mAb #61193

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous Rabbit IgG
IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using MUC5AC (E3O9I) XP® Rabbit mAb.

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IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using MUC5AC (E3O9I) XP® Rabbit mAb.

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IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded A549 cell pellet (left, high-expressing) or HeLa cell pellet (right, low-expressing) using MUC5AC (E3O9I) XP® Rabbit mAb.

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IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using MUC5AC (E3O9I) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

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IF-IC

Confocal immunofluorescent analysis of A549 cells (left, positive) and HeLa cells (right, negative) using MUC5AC (E3O9I) Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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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

3.

Peroxide Blocking

Peroxide Block*

5 min.

 

WASH

BOND Wash Solution

3x 0:00 min.

4.

Protein Block

CST #5425 NGS or #15019 Animal-Free Blocking Solution

20 min.

5.

Primary Antibody

Dilute in #8112 Antibody Diluent

30 min.

 

WASH

BOND Wash Solution

3x 2:00 min.

6.

Secondary Detection

Novolink Polymer*

10 min.

 

WASH

BOND Wash Solution/Deionized Water

Custom (see below)

7a.

Visualization

Mixed DAB Refine*

0:00 min.

7b.

Visualization

Mixed DAB Refine*

10 min.

 

WASH

Deionized Water

3x 0:00 min.

8.

Counterstain

Hematoxylin*

5 min.

 

WASH

Deionized Water

3x 0:00 min.

 

WASH

BOND Wash Solution

3x 0:00 min.

 

WASH

Deionized Water

3x 0:00 min.

9.

Dehydration (Offline):
Incubate sections in 95% ethanol two times for 10 sec. each.
Repeat in 100% ethanol, incubating sections two times for 10 sec each.
Repeat in xylene, incubating sections two times for 10 sec each.
Mount sections with coverslips and mounting medium (#14177).

  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 February 2017

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).

posted February 2010

revised March 2016

Protocol Id: 283

Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified 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. Methanol, 100%
  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 / 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:

  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 ice-cold 100% methanol.
  2. Allow cells to fix for 15 minutes at -20°C.
  3. Aspirate fixative, 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 minutes.
  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 minutes each.
  6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hours at room temperature in dark.
  7. Rinse in 1X PBS as in step 5.
  8. Coverslip slides with Prolong® Gold Antifade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
  9. For best results, examine specimens immediately using appropriate excitation wavelength. For long-term storage, store slides flat at 4°C protected from light.

posted December 2010

Protocol Id: 3

Application Dilutions
IHC-Leica® Bond™ 1:400
Immunohistochemistry (Paraffin) 1:400
Immunofluorescence (Immunocytochemistry) 1:400
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.

MUC5AC (E3O9I) XP® Rabbit mAb recognizes endogenous levels of total MUC5AC protein.

Species Reactivity:

Human

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro2712 of human MUC5AC protein.

Mucins are a family of macromolecules that line and protect the respiratory epithelium from microbes and pollutants in the local environment. Of the family members that are known to date, some are produced in a cell type and tissue-specific manner, suggesting distinct biological roles for members. Some members polymerize after secretion to form gel-like substances that coat the epithelial layer (1). MUC16 is a member of the mucin family, which are a high molecular weight, O-glycosylatyed proteins that play an important role in forming mucous barrier (2) and are found on the apical surface of the epithelia. It contains an extracellular domain at its amino acid terminus, a large tandem repeat and a transmembrane domain with a short cytoplasmic domain. MUC16 has been linked to lung cancer (3), and ovarian cancer (4). It provides protective, lubricating barrier against particles and infectious agents at mucosal surfaces (5).

  1. Ma, J. et al. (2018) Chest 154, 169-176.
  2. Argüeso, P. et al. (2003) Invest Ophthalmol Vis Sci 44, 2487-95.
  3. Kanwal, M. et al. (2018) Oncotarget 9, 12226-12239.
  4. Williams, K.A. et al. (2014) PLoS One 9, e88334.
  5. Gilowska, I. (2014) Postepy Hig Med Dosw (Online) 68, 842-50.
Entrez-Gene Id
4586
Swiss-Prot Acc.
P98088
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SignalStain is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.

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