Dp12 ccd camera

Immunostaining was applied on paraffin and cryostat sections. Paraffin sections were treated by microwave for 20 min at 400W in citrate buffer (pH 6.0) after deparaffinization and hydratation. Five μm thick frozen sections cut from each tissue were fixed in acetone for 10 min, air-dried at room temperature for 1 hour. After antigen retrieval both frozen and paraffin samples were incubated for 1 hour at room temperature with primary antibody: NCAM clone Eric-1 (1:100, Ancell Corporation, USA), NCAM clone 123C3.D5 (LabVision, USA), FGFR1 clone M19B2 (1:100, Abcam, UK), HE4 (1:40, ab24480, Abcam, UK), integrin α5β1 clone SAM-1 (1:200, Chemicon Europe). NCAM/Eric-1 was used on cryostat samples, while paraffin samples were incubated with NCAM/123C3.D5. The EnVision staining method (DAKO, Denmark), visualization of antigen-antibody reaction by 3,3'-diaminobenzidine (DAB) and subsequent counterstaining with hemalaun (Merck, USA), were conducted. Controls were performed as previously described [2 (link)], and for mouse monoclonal antibodies as isotype control mouse IgG1 (ab91353, Abcam, UK) antibody was also used. Slides were evaluated using the light microscope BX53 with DP12 CCD camera (Olympus, Germany).

Immunostaining was applied on 5 μm thick paraffin sections. After deparaffinization and rehydration, the sections were treated by microwave for 20 min at 400 W in citrate buffer (pH 6.0). After antigen retrieval, samples were incubated for 1 hour at room temperature with primary antibody for nestin (Santa Cruz, USA, sc-23927, dilution 1:100). Sections were then treated with EnVisionTM Detection System (DAKO, Germany) using 3-amino-9-ethylcarbazole (AEC) as substrate, and counterstained with hematoxylin. Negative controls were performed by omitting the first antibody and stained by the EnVisionTM method, and for mouse monoclonal antibodies as isotype control mouse IgG1 (ab91353, Abcam, UK) antibody was also used. The slides were evaluated using a light microscope BX53 with DP12-CCD camera (Olympus, Germany).