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71299
F4/80 (BM8.1) Rat mAb
Primary Antibodies

F4/80 (BM8.1) Rat mAb #71299

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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 F4/80 (BM8.1) Rat mAb (green). 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) 1:100
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.

F4/80 (BM8.1) Rat mAb recognizes endogenous levels of total F4/80 protein. This antibody detects an epitope within the extracellular domain.

Species Reactivity:

Mouse

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

F4/80 (EMR1) is a heavily glycosylated G-protein-coupled receptor and is a well-established marker for mouse macrophages (1-3). Expression of F4/80 has also been observed in microglia and subset populations of dendritic cells (4).

  1. Hirsch, S. et al. (1981) J Exp Med 154, 713-25.
  2. Austyn, J.M. and Gordon, S. (1981) Eur J Immunol 11, 805-15.
  3. McKnight, A.J. et al. (1996) J Biol Chem 271, 486-9.
  4. Greter, M. et al. (2015) Front Immunol 6, 249.
Entrez-Gene Id
13733
Swiss-Prot Acc.
Q61549
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
ProLong is a registered trademark of Life Technologies Corporation.

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