Immunohistochemical staining of ubiquitin in a 7 months-old MPS IIIA mouse brain (PFA fixed) using ab7254.

Sections, 40 µm thick, were cut sagittally on a vibratome. Slices were permeabilized in 1% triton X-100/PBS for 1 hour at room temperature. The samples were placed on vectabond-coated glass slides and dried overnight. They were then rehydrated in PBS and washed in ice-cold methanol for 10 min.

The slices were washed in 0.3% H₂O₂ in methanol at room temperature and incubated sequentially with 0.5% normal donkey serum, ab7254 and biotin-labeled F(ab')₂ donkey secondary antibody in Tris-buffered saline (pH 7.5) containing 2% BSA and 0.02% Tween 20. The signal was detected with an ABC kit, visualized with diaminobenzidine and counterstained with hematoxylin.

All lanes : Anti-Ubiquitin antibody [Ubi-1] (ab7254) at 1/100 dilution

Lane 1 : HeLa cell lysate
Lane 2 : HeLa cell lysate with MG132
Lane 3 : Mouse brain tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Anti mouse IgG at 1/10000 dilution

IHC image of ab7254 staining in mouse alzheimer brain formalin fixed paraffin embedded tissue section, using MOM detection kit, ab127055. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was incubated with ab7254, 5µg/ml, for 15 mins at room temperature.  DAB was used as the chromogen (ab103723). The section was counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Human normal placenta (ab29745). Staining is localised in the cytoplasm and in the nuclei. Left panel: with primary antibody at 1 ug/ml. Right panel: isotype control. Sections were stained using an automated system DAKO Autostainer Plus, at room temperature: sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffers citrate pH6.1 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for mouse for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

HeLa cells were co-transfected with a plasmid expressing a target protein together with Ubi expressing vector for 24 hours and either left untreated (Contr) or were treated with 10 µM MG-132 for 6 hours (+MG132). Then the protein of interest was pulled down using Flag agarose beads and and probed with ab7254 at a 1/2000 dilution. The secondary used was an Alexa-Fluor 680 conjugated goat anti-mouse polyclonal used at a 1/10000 dilution. The protein is known to be degraded through proteasome.

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