This is a knockout-validated antibody summary, based on the publication "Biochemical and morphological detection of inclusion bodies in autophagy-deficient mice", as cited below [1]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

Guinea pig polyclonal

Company: MBL

Antibody: p62

Catalog number: PM045

Summary: Rabbit polyclonal IgG against human p62 protein( aa 120-440). Reacts with human, mouse, hamster and rat protein. Suitable for western blot, immunoprecipitation, immunocytochemistry and immunohistochemistry

Western blot | Immunohistochemistry

WB: Liver lysate from WT, Atg7F/F:Mx1 and p62-deficient mice. Tissue was homogenized in buffer consisting of 0.25 M sucrose, 10 mM 2-[4- (2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES), pH 7.4, and 1 mM dithiothreitol (DTT).

IHC: Brain and liver tissues were immersed in OCT/sucrose solution and then frozen in isopentane with dry ice. Cryosections were cut into 10-mm-thick sections with a cryostat (CM3050S, Leica, Nussloch, Germany).

WB: BlockAce (Dainippon Pharmaceutical, Osaka, Japan, #UK-B80) for 30 min at room temperature (RT).

IHC: f 0.01 M PB, pH 7.2, 0.15 M NaCl, 0.05% Tween 20, and 1% normal goat serum (NGS, Vector Laboratories, Burlingame, CA, #S-1000) for 20 min at RT.

WB: 1:500 dilution for 16 h at 4 C.

IHC: 1:400–800 in blocking solution for 2–3 days at 4 C or 1 h at RT in a humidified chamber.

WB: horseradish peroxidase (HRP)–conjugated secondary antibodies ( Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, #106-035-063 for anti–guinea pig IgG) for 30 min at RT.

IHC: donkey anti-guinea pig IgG conjugated with HRP (Chemicon, Temecula, CA, #AP193P) for 1 h at RT.

WB: enhanced chemiluminescence (Western Lightning, Perkin Elmer).

IHC: Incubate in 0.05 M Tris-HCl buffer, pH 7.6, containing 0.0125% diaminobenzidine (DAB, Dojindo Laboratories, Kumamoto, Japan, #349-0093) and 0.002% H2O2 for 5–10 min. Immerse successively in 70, 80, 90, 95, 100, and 100% alcohol for 5–10 min each for dehydration, and finally in xylene twice for 5–10 min each, and mount with Canada balsam (Wako, #034-01042). View with a Nomarski differential interference contrast microscope (Olympus, Tokyo).

Please see Figures 9.1 (WB) and 9.2 (IHC) in the article [1].

Unlike for brain tissue immunohistochemistry, the antibody seems to be nonspecific in liver tissue. Thus, caution should be taken when the aim of the study is evaluation of cytoplasmic diffuse staining instead of the detection of inclusions.

If the antibody described in this summary is a polyclonal antibody, since polyclonal antibodies are of limited quantity, please inquire the supplier whether any current polyclonal antibody with the same catalog number is exactly the same as the one described in this summary. Sometimes, different bleeds or different animals are used, usually with a different lot number. In such cases, the result in this summary may not apply to the new antibody with the same catalog number.