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86285
MHC Class II (I-A/I-E) (M5/114.15.2) Rat mAb
Primary Antibodies
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MHC Class II (I-A/I-E) (M5/114.15.2) Rat mAb #86285

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Confocal immunofluorescent analysis of barrier associated macrophages in fixed frozen mouse brain labeled with MHC Class II (I-A/I-E) (M5/114.15.2) Rat mAb (left, green), Iba1/AIF-1 (E4O4W) XP® Rabbit mAb #17198 (right, red), and DAPI #4083 (right, blue).
Confocal immunofluorescent analysis of fixed frozen mouse lung labeled with MHC Class II (I-A/I-E) (M5/114.15.2) Rat mAb (left, green), Iba1/AIF-1 (E4O4W) XP® Rabbit mAb #17198 (right, red), DyLight 650 Phalloidin #12956 (right, gray pseudocolor), and DAPI #4083 (right, blue).
Confocal immunofluorescent analysis of fixed frozen mouse kidney labeled with MHC Class II (I-A/I-E) (M5/114.15.2) Rat mAb (left, green), Iba1/AIF-1 (E4O4W) XP® Rabbit mAb #17198 (right, red), DyLight 650 Phalloidin #12956 (right, gray pseudocolor), and DAPI #4083 (right, blue).
Confocal immunofluorescent analysis of fixed frozen mouse colon labeled with MHC Class II (I-A/I-E) (M5/114.15.2) Rat mAb (left, green), Iba1/AIF-1 (E4O4W) XP® Rabbit mAb #17198 (right, red), DyLight 650 Phalloidin #12956 (right, gray pseudocolor), and DAPI #4083 (right, blue).
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To Purchase # 86285
Cat. # Size Qty. Price
86285S		 100 µl

Carrier-Free Bulk & Custom Formulation

Supporting Data

REACTIVITY M
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Rat IgG2b 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
Immunofluorescence (Frozen) 1:50 - 1:200

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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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-Rat secondary antibodies:

  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: 152

Specificity / Sensitivity

MHC Class II (I-A/I-E) (M5/114.15.2) Rat mAb recognizes endogenous levels of total MHC class II (I-A/I-E) proteins. This antibody detects epitopes within the extracellular domain of MHC class II (I-A/I-E).

Species Reactivity:

Mouse

Source / Purification

This monoclonal antibody was purified from tissue culture supernatant via affinity chromatography.

Background

Major histocompatibility complex class II (MHC class II) molecules are heterodimeric, transmembrane glycoproteins expressed on the surface of antigen-presenting cells, such as macrophages, dendritic cells, and B cells. Expression can also be induced on other cell types through interferon-γ signaling (1). Prior to being displayed on the cell membrane, MHC class II molecules are loaded with exogenous peptide antigens approximately 15-24 amino acids in length that were derived from endocytosed extracellular proteins digested in the lysosome (2). Antigen-presentation through MHC class II is required for T cell activation during the immune response to extracellular pathogens (2). In humans, the MHC class II protein complex is encoded by the human leukocyte antigen gene complex (HLA). HLAs corresponding to MHC class II are HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR (3).

The M5/114.15.2 antibody reacts with mouse MHC class II, both I-A and I-E subregion-encoded glycoproteins (I-Ab, I-Ad, I-Aq, I-Ed, I-Ek, not I-Af, I-Ak, or I-As). It detects a polymorphic determinant present on B cells, monocytes, macrophages, dendritic cells, and activated T lymphocytes from mice carrying the H-2b, H-2d, H-2q, H-2p, H-2r and H-2u haplotypes, but not from mice carrying the H-2s or H-2f haplotypes (4-7). The M5/114 mAb is reported to inhibit I-A-restricted T cell responses of the H-2b, H-2d, H-2q, H-2u but not H-2f, H-2k, or H-2s haplotypes (8,9).

Limited Uses

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For Research Use Only. Not for Use in Diagnostic Procedures.
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