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64095
S100A1 (4C4.9) Mouse mAb
Primary Antibodies
Monoclonal Antibody
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S100A1 (4C4.9) Mouse mAb #64095

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  1. IHC
Immunohistochemical analysis of paraffin-embedded human melanoma using S100A1 (4C4.9) Mouse mAb performed on the Leica® BOND™ Rx.
Immunohistochemical analysis of paraffin-embedded normal human brain using S100A1 (4C4.9) Mouse mAb.
Immunohistochemical analysis of paraffin-embedded normal human breast using S100A1 (4C4.9) Mouse mAb.
Immunohistochemical analysis of paraffin-embedded human melanoma using S100A1 (4C4.9) Mouse mAb. Note the use of the SignalStain® Vivid Purple Peroxidase Substrate Kit #96632.
Immunohistochemical analysis of paraffin-embedded human granulosa cell tumor of the ovary using S100A1 (4C4.9) Mouse mAb (left) compared to concentration-matched Mouse (E5Y6Q) mAb IgG2a Isotype Control #61656 (right).
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To Purchase # 64095
Cat. # Size Qty. Price
64095S		 100 µl

Carrier-Free Bulk & Custom Formulation

Supporting Data

REACTIVITY H
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Mouse IgG2a kappa

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 1:800 - 1:3200
Immunohistochemistry (Paraffin) 1:400 - 1:1600

Storage

Supplied in a PBS-based buffer with ~1% BSA and less than 0.1% sodium azide. Stable for 6 months when stored at 4°C. Do not aliquot the antibody.

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 Polymer 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.
6 Post Primary Mouse Linker Polymer Refine Detection Kit Post Primary*  10 min. 
  WASH BOND™ Wash Solution  3x 2:00 min.
7 Secondary Detection Polymer Refine Detection Kit Polymer*  10 min. 
  WASH BOND™ Wash Solution/Deionized Water Custom (see below)
8a Visualization Polymer Refine Detection Kit Mixed DAB Refine*  0:00 min. 
8b Visualization Polymer Refine Detection Kit Mixed DAB Refine*  10 min. 
  WASH Deionized Water 3x 0:00 min.
9 Counterstain Polymer 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. 
10 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.  
11 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: 1445

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, Mouse #8125).
  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, Mouse #8125) 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, Mouse #8125) 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, Mouse) #8125 SignalStain® Boost IHC Detection Reagent (AP, Mouse) #31926
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: 280

Specificity / Sensitivity

S100A1 (4C4.9) Mouse mAb recognizes endogenous levels of S100A1 protein.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a purified bovine brain S100A1 protein.

Background

Despite their relatively small size (8-12 kDa) and uncomplicated architecture, S100 proteins regulate a variety of cellular processes, such as cell growth and motility, cell cycle progression, transcription, and differentiation. To date, 25 members have been identified, including S100A1-S100A18, trichohyalin, filaggrin, repetin, S100P, and S100Z, making it the largest group in the EF-hand, calcium-binding protein family. Interestingly, 14 S100 genes are clustered on human chromosome 1q21, a region of genomic instability. Research studies have demonstrated that significant correlation exists between aberrant S100 protein expression and cancer progression. S100 proteins primarily mediate immune responses in various tissue types but are also involved in neuronal development (1-4).

Each S100 monomer bears two EF-hand motifs and can bind up to two molecules of calcium (or other divalent cation in some instances). Structural evidence shows that S100 proteins form antiparallel homo- or heterodimers that coordinate binding partner proximity in a calcium-dependent (and sometimes calcium-independent) manner. Although structurally and functionally similar, individual members show restricted tissue distribution, are localized in specific cellular compartments, and display unique protein binding partners, which suggests that each plays a specific role in various signaling pathways. In addition to an intracellular role, some S100 proteins have been shown to act as receptors for extracellular ligands or are secreted and exhibit cytokine-like activities (1-4).

S100A1 is abundantly expressed in cardiac and skeletal muscle where it plays a major role in regulating calcium-dependent contractility (5,6). S100A1 and calmodulin bind and differentially regulate ryanodine receptors (RyRs), thereby modulating skeletal and cardiac muscle function (7). In addition to RyRs (RyR1 and RyR2), S100A1 has also been shown to interact with other components of the calcium-dependent cardiac signaling cascade, including SERCA2a and phospholamban (8). Studies in animal models strongly suggest that S100A1 plays a significant role in the development of heart failure (1). In non-cardiac tissues, S100A1 has been shown to regulate cytoskeletal signaling, neurotransmitter release, enzymatic activity, transcription factors, and other calcium-binding proteins via direct interaction or via regulation of scaffolding and signaling components in each pathway (4).

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.
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