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24090
β-Amyloid (1-42) (E6D5M) Rabbit mAb
Primary Antibodies
Monoclonal Antibody

β-Amyloid (1-42) (E6D5M) Rabbit mAb #24090

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  1. IHC
  2. IF
Immunohistochemistry Image 1: β-Amyloid (1-42) (E6D5M) Rabbit mAb
Immunohistochemical analysis of paraffin-embedded human Alzheimer's brain using β-Amyloid (1-42) (E6D5M) Rabbit mAb.
Immunohistochemistry Image 2: β-Amyloid (1-42) (E6D5M) Rabbit mAb
Immunohistochemical analysis of paraffin-embedded human Alzheimer's brain using β-Amyloid (1-42) (E6D5M) Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right). 
Immunohistochemistry Image 3: β-Amyloid (1-42) (E6D5M) Rabbit mAb
Immunohistochemical analysis of paraffin-embedded human Alzheimer's brain (left) or normal brain (right) using β-Amyloid (1-42) (E6D5M) Rabbit mAb.
Immunohistochemistry Image 4: β-Amyloid (1-42) (E6D5M) Rabbit mAb
Immunohistochemical analysis of paraffin-embedded Tg2576 mouse brain using β-Amyloid (1-42) (E6D5M) Rabbit mAb. Note: Tg2576 is a mouse model of Alzheimer's disease expressing mutant human APP with the double mutation K670N, M671L (Swedish mutation).
Immunohistochemistry Image 5: β-Amyloid (1-42) (E6D5M) Rabbit mAb
Immunohistochemical analysis of paraffin-embedded Tg2576 mouse brain (left) or wild-type mouse brain (right) using β-Amyloid (1-42) (E6D5M) Rabbit mAb. Note: Tg2576 is a mouse model of Alzheimer's disease expressing mutant human APP with the double mutation K670N, M671L (Swedish mutation).
Immunofluorescence Image 1: β-Amyloid (1-42) (E6D5M) Rabbit mAb
Confocal immunofluorescent analysis of brain from the 5XFAD mouse model of Alzheimer’s disease using Beta-APP42 (E6D5M) Rabbit mAb (green) and GFAP (GA5) Mouse mAb (Alexa Fluor® 555 Conjugate) #3656 (red). Sections were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
To Purchase # 24090
Cat. # Size Qty. Price
24090S
100 µl $ 276

Supporting Data

REACTIVITY H
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Rabbit IgG

Application Key:

  • WB-Western Blot
  • 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
  • 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
  • All-All Species Expected

Product Usage Information

Application Dilution
Immunohistochemistry (Paraffin) 1:200 - 1:800
Immunofluorescence (Frozen) 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.

Protocol

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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  

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 (Frozen)

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) to 9.5 ml 1X PBS) and 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:

    NOTE: When using any primary or fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to determine which dilution allows for the strongest specific signal with the least background for your sample.

  6. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Frozen/Cryostat Sections (IF-F)

  1. For fixed frozen tissue proceed with Immunostaining (Section C).
  2. For fresh, unfixed frozen tissue, please fix immediately, as follows:
    1. Cover sections with 4% formaldehyde dilute in 1X PBS.

      NOTE: Formaldehyde is toxic, use only in fume hood.

    2. Allow sections to fix for 15 minutes at room temperature.
    3. Aspirate liquid, 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 min.
  2. While blocking, prepare primary antibody by diluting as indicated on protocol 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 November 2006

revised July 2016

Protocol Id: 151

Specificity / Sensitivity

β-Amyloid (1-42) (E6D5M) Rabbit mAb recognizes endogenous levels of total Aβ-42 protein. This antibody does not cross-react with other β-amyloid peptides.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human β-amyloid (1-42) peptide.

Background

Amyloid β (Aβ) precursor protein (APP) is a 100-140 kDa transmembrane glycoprotein that exists as several isoforms (1). The amino acid sequence of APP contains the amyloid domain, which can be released by a two-step proteolytic cleavage (1). The extracellular deposition and accumulation of the released Aβ fragments form the main components of amyloid plaques in Alzheimer's disease (1). APP can be phosphorylated at several sites, which may affect the proteolytic processing and secretion of this protein (2-5). Phosphorylation at Thr668 (a position corresponding to the APP695 isoform) by cyclin-dependent kinase is cell-cycle dependent and peaks during G2/M phase (4). APP phosphorylated at Thr668 exists in adult rat brain and correlates with cultured neuronal differentiation (5,6).
  1. Selkoe, D.J. (1996) J Biol Chem 271, 18295-8.
  2. Caporaso, G.L. et al. (1992) Proc Natl Acad Sci USA 89, 3055-9.
  3. Hung, A.Y. and Selkoe, D.J. (1994) EMBO J 13, 534-42.
  4. Suzuki, T. et al. (1994) EMBO J 13, 1114-22.
  5. Ando, K. et al. (1999) J Neurosci 19, 4421-7.
  6. Iijima, K. et al. (2000) J Neurochem 75, 1085-91.

Pathways & Proteins

Explore pathways + proteins related to this product.

Limited Uses

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For Research Use Only. Not For Use In Diagnostic Procedures.
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