Editor's Note: All antibody reviews appear in protocol format.
Primary Antibody: Pericentrin Polyclonal Antibody
Antigen/Epitope: Pericentrin
Supplier and catalog number: Covance PRB-432C
Sample: 30-50 in vivo matured mouse oocytes
Secondary Antibody: Alexa-Fluor 568 goat anti-mouse IgG
Supplier and catalog number: Life Technologies A-11004 (from Molecular Probes)
Block: 2% BSA, 2% Powdered Milk, 2% Normal Goat Serum in 0.1 M Glycine and 0.01% Triton X-100
Incubation time: Overnight
Incubation temperature: 4°C
Primary Antibody Incubation
Dilution used: 1/100
Diluent composition: 2% BSA, 2% Powdered Milk, 2% Normal Goat Serum in 0.1 M Glycine and 0.01% Triton X-100
Incubation time: 90 min
Incubation temperature: 37°C
Secondary Antibody Incubation
Dilution used: 1/800
Diluent composition: 2% BSA, 2% Powdered Milk, 2% Normal Goat Serum in 0.1 M Glycine and 0.01% Triton X-100
Incubation time: 1 hr
Incubation temperature: 37°C
Detection Method: Zeiss IM-35 inverted microscope using 50-W mercury arc lamp and a 40x Neofluor objective. Digital images were collected with a Hamamatsu Orca ER digital camera (model #C4742-95) interfaced with a MetaMorph Imaging System (Molecular Devices, Sunnyvale, CA).
Results Summary: The image below shows M-II spindles of mouse oocytes. (Blue shows chromatin staining used during the mounting procedure.)
Additional notes/tips: As with Gamma-Tubulin, Pericentrin needs a strong and "clean" fixation in order to show up. Therefore, I use Triton X-100 in the fixative to wash out some of the cytoplasmic content evidencing the gamma-tubulin protein.
(Note: The image at the top of this page is from Covance.)