Qimaging retiga 2000r fast 1394 camera

We assessed expression pattern of extracellular matrix proteins (type-II collagen, aggrecan, and cartilage oligomeric matrix protein, that is, COMP) and CD44 (a cell surface antigen) through immunostaining as described.^{4 (link)} The primary antibodies used are shown in Table 1. Images were captured on an Eclipse E800 microscope (Nikon, Tokyo, Japan) with QImaging Retiga 2000R Fast 1394 camera and MetaMorph software v7.7 (Molecular Devices, Sunnyvale, CA).

Sections were deparaffinized, rehydrated and digested with proteinase K (10 μg/ml for 20 min at 37°C) to enhance antibody penetration and then blocked with 10% serum in PBS for 1 h at room temperature. Next, the slides were incubated with the IIF antibody against the type II collagen triple helical domain (15 (link)) (1:100) at 4°C overnight in the blocking solution, washed and incubated with a second primary antibody against either aggrecan (LEC7, G3 domain of aggrecan, a gift from Dr. Kurt Doege, Louisiana State University, (16 (link)) (1:100), type I collagen (Abcam, USA) (1:200), or COMP (gift from Dr. Dick Heinegard, University of Lund, Sweden) (1:100). Fluorescent detection of each protein was achieved using either secondary goat Alexa Fluor 488-conjugated anti-rabbit IgG (1:250) or goat Alexa Fluor 594-conjugated anti-rat IgG antibodies (1:250) (Invitrogen, USA), and counterstained with DAPI (1:1000)(Vector Laboratories, Burlingame, CA). The images were captured using a 20× objective on an Eclipse E800 microscope (Nikon), Q Imaging Retiga 2000R Fast 1394 camera and MetaMorph software v 7.7 (Molecular Devices, Sunnyvale, CA).