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58970
Iba1/AIF-1 (E5N4J) Mouse mAb (IHC Formulated)
Primary Antibodies

Iba1/AIF-1 (E5N4J) Mouse mAb (IHC Formulated) #58970

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  1. IHC

Immunohistochemical analysis of paraffin-embedded human neuroendocrine carcinoma of the lung using Iba1/AIF-1 (E5N4J) Mouse mAb (IHC Formulated) performed on the Leica® BOND™ Rx.

Immunohistochemical analysis of paraffin-embedded human ovarian clear cell carcinoma using Iba1/AIF-1 (E5N4J) Mouse mAb (IHC Formulated) (left) or Iba1/AIF-1 (E4O4W) XP® Rabbit mAb #17198 (right) performed on the Leica® BOND™ Rx. These two antibodies detect independent, unique epitopes on human Iba1/AIF-1 protein. The similar staining patterns obtained with both antibodies help to confirm the specificity of the staining.

Immunohistochemical analysis of paraffin-embedded normal human brain using Iba1/AIF-1 (E5N4J) Mouse mAb (IHC Formulated).

Immunohistochemical analysis of paraffin-embedded THP-1 cell pellet (left, positive) or SH-SY5Y cell pellet (right, negative) using Iba1/AIF-1 (E5N4J) Mouse mAb (IHC Formulated).

Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma using Iba1/AIF-1 (E5N4J) Mouse mAb (IHC Formulated) (left) compared to concentration-matched Mouse (G3A1) mAb IgG1 Isotype Control #5415 (right).

Immunohistochemical analysis of paraffin-embedded human mucoepidermoid carcinoma of the larynx using Iba1/AIF-1 (E5N4J) Mouse mAb (IHC Formulated).

Immunohistochemical analysis of paraffin-embedded human non-Hodgkin's lymphoma using Iba1/AIF-1 (E5N4J) Mouse mAb (IHC Formulated) performed on the Leica® BOND™ Rx.

To Purchase # 58970S
Product # Size Price
58970S
100 µl N/A

Supporting Data

REACTIVITY H
SENSITIVITY Endogenous
MW (kDa)
Isotype Mouse IgG1, 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 Dilutions
IHC-Leica® Bond™ 1:100 - 1:400
Immunohistochemistry (Paraffin) 1:100 - 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.

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).

posted February 2010

revised March 2016

Protocol Id: 280

Specificity / Sensitivity

Iba1/AIF-1 (E5N4J) Mouse mAb (IHC Formulated) recognizes endogenous levels of total Iba1/AIF-1 protein.

Species Reactivity:

Human

Species predicted to react based on 100% sequence homology:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu32 of human Iba1/AIF-1 protein.

Background

Ionized calcium-binding adaptor molecule 1 (Iba1), also known as allograft inflammatory factor 1 (AIF-1), is an evolutionarily conserved cytoplasmic calcium binding protein containing a central pair of EF-hand calcium binding motifs (1,2). Iba1/AIF-1 was originally cloned from activated macrophages in human atherosclerotic allogenic heart grafts undergoing chronic transplant rejection as well as from rat monocytes (3,4). Its function is not very well understood, but Iba1/AIF-1 expression is upregulated in response to interferon-gamma and, therefore, could modulate macrophage-dependent immune response (3). As an F-actin-binding protein, Iba1/AIF-1 may function to remodel the actin cytoskeleton and contribute to morphological changes that correlate with various microglial/macrophage states (5). Iba1/AIF-1 is also uniquely expressed in cells of monocytic lineage and is, therefore, widely used as a marker for microglia/macrophages in the brain and other tissue (6).

  1. Schulze, J.O. et al. (2008) FEBS J 275, 4627-40.
  2. Deininger, M.H. et al. (2002) FEBS Lett 514, 115-21.
  3. Utans, U. et al. (1995) J Clin Invest 95, 2954-62.
  4. Imai, Y. et al. (1996) Biochem Biophys Res Commun 224, 855-62.
  5. Kanazawa, H. et al. (2002) J Biol Chem 277, 20026-32.
  6. Imai, Y. et al. (1996) Biochem Biophys Res Commun 224, 855-62.

Pathways & Proteins

Explore pathways + proteins related to this product.

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.
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.
LEICA is a registered trade​mark of Leica Microsystems IR GmbH.