CEBP Beta antibody | knockout validation | Abcam ab32358

This is a knockout-validated antibody summary, based on the publication "CCAAT/enhancer binding protein β is required for satellite cell self-renewal", as cited below [1]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

CEBP Beta antibody | knockout validation | Abcam ab32358 figure 1
Figure 1. Western analysis of C/EBPβ expression in satellite cells isolated from the hindlimb of WT and C/EBPβ−/− mice injected with tamoxifen for 5 days.
Antibody information

Rabbit monoclonal IgG

Company: Abcam

Antibody: CEBP Beta

Catalog number: ab32358

Summary: Rabbit monoclonal IgG against a synthetic peptide corresponding to residues in the C-terminus of rat CEBPB. Reacts with mouse, rat, and human. Suitable for western blot, immunoprecipitation, immunocytochemistry/immunofluorescence, flow cytometry, and EMSA. Unsuitable for immunohistochemistry (paraffin). The antibody could cross-react with CEBP epsilon (32, 27 and 14 kDa, 82% homology) and CEBP alpha (42 and 30 kDa, 73% homology) in human, mouse and rat.

CEBP Beta antibody | knockout validation | Abcam ab32358 figure 2
Figure 2. Cross-sections of uninjured tibialis anterior were immunostained for Pax7 (red), C/EBPβ (green), and laminin (white). Nuclei were counterstained with DAPI (blue). Representative pictures are shown. Scale bar, 20 μm. From [1].
Validation Method

Western blot | Immunohistochemistry

Sample

Primary myoblasts and tibialis anterior from C/EBPβfl/fl and C/EBPβ−/− Pax7CreER−/+ mice. WB: Whole-cell extracts were prepared from primary myoblasts. IHC: left tibialis anterior muscle was collected, embedded in Tissue-Tek OCT compound, flash frozen in isopentane cooled by liquid nitrogen, and sectioned (8-μm thick) for immunofluorescence. Cryosections were thawed at room temperature, fixed in 4% PFA, and processed for antigen retrieval in citrate buffer at 95 °C for 20 min.

Blocking agent

IHC: PBS containing 0.1% Triton X-100 and 5% donkey serum (Cedarlane).

Primary incubation

IHC: overnight at 4 °C.

Secondary incubation

IHC: secondary antibodies conjugated to a fluorescent dye (Cy3, Alexa 488, or Alexa Fluor 647) from Jackson ImmunoResearch.

Detection

WB: Chemiluminescence was detected with the ChemiDocTM MP System (Bio-Rad Laboratories).

IHC: Digital images of the stained myoblasts, myofibers, and muscle sections were acquired at room temperature using a microscope (Leica DM 3000B), Infinity-3 camera (Lumenera), and Infinity Capture imaging software (Lumenera).

Clone note

The same clone (E299) is sold as Abcam ab32358; EMD Millipore 04-1153.

References
  1. Lala-Tabbert N, AlSudais H, Marchildon F, Fu D, Wiper-Bergeron N. CCAAT/enhancer binding protein β is required for satellite cell self-renewal. Skelet Muscle. 2016;6:40 pubmed