Rabbit anti p16ink4a

Paraffin-embedded tissue sections (7–10 μm) were deparaffinized and rehydrated. Antigens were unmasked using heat-mediated citrate buffer. Double immunofluorescent studies were carried out by blocking the sections with CAS-BLOCK (Invitrogen Corporation) or 10% donkey serum for 1 hr at room temperature, followed by sequential incubation with a goat anti-laminin γ2 (LCC, 1;100, Santa Cruz Biotechnology, Inc.), and either rabbit anti-p16^INK4a (1:30, Santa Cruz Biotechnology, Inc.), or rabbit polyclonal anti-laminin γ2 (L4, a gift from Dr. Takako Sasaki) (Sasaki et al., 2001 (link)), diluted in PBS/0.05% Tween-20/1.5% Normal Donkey Serum (NDS) or appropriate IgG controls. After overnight incubation at 4°C, slides were treated with appropriate secondary antibodies (donkey anti-goat Alexa Fluor 568 or donkey anti-rabbit Alexa Fluor 488 1:1,000, Invitrogen) for 1 hr at room temperature in the dark. Sections were counterstained with DAPI and mounted in ProLong Gold anti-fade reagent (Invitrogen Corporation). 3D Z-stacks of confocal images were collected using a Zeiss LSM 510 Meta Confocal Microscope equipped with multitrack laser lines to select from UV 405 nm, tunable argon, and two HeNe lasers (594 and 633 nm). Confocal projection images were created to be viewed down into the image stack along the Z-axis.