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46512
CD11b/ITGAM (M1/70) Rat mAb
Primary Antibodies

CD11b/ITGAM (M1/70) Rat mAb #46512

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
M Endogenous Rat IgG2b, kappa
IF-F

Confocal immunofluorescent analysis of mouse brain microglia (left) or peripheral macrophages of the liver (right) using CD11b/ITGAM (M1/70) Rat mAb (green). Sections were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

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IF-F

Confocal immunofluorescent analysis of brain from the 5XFAD mouse model of Alzheimer’s disease using CD11b/ITGAM (M1/70) Rat mAb (green) and β-Amyloid (D54D2) XP® Rabbit mAb #8243 (red). Sections were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

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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 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 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

Application Dilutions
Immunofluorescence (Frozen) 1:50
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.

CD11b/ITGAM (M1/70) Rat mAb recognizes endogenous levels of total CD11b/ITGAM protein. This antibody detects an epitope within the extracellular domain.

Species Reactivity:

Mouse

Species predicted to react based on 100% sequence homology:

Human

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

Cluster of differentiation molecule 11b (CD11b)/Integrin alpha M (ITGAM) is a transmembrane protein forming heterodimers that are composed of α and β subunits (1). CD11b is expressed by, and commonly used as a marker for, myeloid lineage cells, including neutrophils, monocytes, macrophages, and microglia (2). CD11b is phosphorylated at Ser1126 (cytoplasmic tail) in neutrophils. Research has shown that this phosphorylation event plays a role for leukocytes traveling from the blood stream to tissues (3). Furthermore, genome-wide association studies have linked CD11b to autoimmune diseases, such as systemic lupus erythematous (SLE) (4).

  1. Solovjov, D.A. et al. (2005) J Biol Chem 280, 1336-45.
  2. Murray, P.J. and Wynn, T.A. (2011) Nat Rev Immunol 11, 723-37.
  3. Fagerholm, S.C. et al. (2006) Blood 108, 3379-86.
  4. Rosetti, F. and Mayadas, T.N. (2016) Immunol Rev 269, 175-93.
Entrez-Gene Id
3684
Swiss-Prot Acc.
P11215
For Research Use Only. Not For Use In Diagnostic Procedures.

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XP is a registered trademark of Cell Signaling Technology, Inc.
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