Ccd camera dfc420

MH3924A cells were cultured on sterile cover glasses (8×8 mm²) for 1 day, then cultured in DMEM with 100 μg/l FK506, or 50 μg/l AMD3100, or 100 μg/l FK506 + 50 μg/l AMD3100. When the cover glasses were covered with cells, the morphologic changes of MH3924A cells were observed with SEM, while a number of cover glasses were used for the immunohistochemical assay to determine the expression of CXCR4. This assay was performed using the anti-CXCR4 antibody (1:100 dilution; Wuhan Boshide Biotechnology Co., Wuhan, China) as the primary antibody and a horseradish peroxidase-conjugated secondary antibody (1:100; DakoCytomation, Glostrup, Denmark). The formed immunocomplex was visualized using the 3,3′-diaminobenzidine (DAB) reagent. The level of CXCR4 was quantified by a computer-assisted image system, which included a Leica CCD camera DFC420 connected to a Leica DM IRE2 microscope (Leica Microsystems, Wetzlar, Germany) (23 (link),24 (link)). Image analysis yielded integrated optical density (IOD) values.

Tissue sections were deparaffinized twice by xylene and then hydrated. Hydrogen peroxide (0.6%) was used to eliminate endogenous peroxidase activity. The sections were blocked with goat serum in Tris-buffered saline for 30 min. Sections were then incubated with anti-VHL antibody (Abcam, 1:100) and anti-Twist1 antibody (Abcam, 1:500) overnight at 4 °C. Secondary antibody was then applied and incubated at 37 °C for 1 hour. Sections were developed with diaminobenzidine and stopped with water. Photographs of representative fields were captured using a Leica CCD camera DFC420 connected to a Leica DMIRE2 microscope (Leica Microsystems Imaging Solutions, Cambridge, UK). Quantification of immunoreactivity was performed on digitally captured color images saved as TIFF files and analyzed using Image-Pro plus 6.0 (Media Cybernetics, Rockville, Maryland, USA). These methods were performed in accordance with the approved guidelines and regulations from the Research Ethics Committee of Huashan Hospital, Fudan University.

The density of positive staining was measured using a computerized image system composed of a Leica CCD camera DFC420 (Leica Microsystems Imaging Solutions, Ltd., Cambridge, UK) connected to a Leica DM4000 B microscope (Leica Microsystems Imaging Solutions, Ltd.). Five representative fields were selected, and consecutive pictures were captured by Leica QWin Plus v3 software under 20 × objective lens (Leica Microsystems Imaging Solutions, N Plan) at a setting identical to the imaging system for analysing. We used the same setting for all slides. The integrated optical density of all ADAMTS-13-, HMGB1-, GR-, Cu/Zn SOD-, and 8-OHdG-positive staining were measured, and the mean ADAMTS-13-, HMGB1-, GR-, Cu/Zn SOD-, and 8-OHdG-positive area/total area was calculated by Leica QWin Plus v3. All images were collected under the same lighting conditions. To avoid observer bias, a blinded investigator quantified all sections. Data were described in terms of mean and standard deviation (mean ± SD) for area %. After calculating the proportion (% pixels) of the stained area to the whole field, each slide's mean (% pixels) staining area was determined. ADAMTS-13, HMGB1, GR, Cu/Zn SOD and 8-OHdG immunohistochemical results were compared between groups using a one-way analysis of variance and Tukey's multiple comparison tests. P < 0.005 was considered statistically significant.

Paraffin-embedded sections of breast or colorectal cancer tissues were deparaffinized at 65 °C for 1–2 h and then subjected to xylene and a graded series of alcohol. Following that, the slides were heated with Sodium citrate-EDTA antigen repair solution (cat. No. P0086, Beyotime) for antigen unmasking. After cooling, the slides were incubated for 1–2 h with primary antibodies at room temperature. The sections were then covered with horseradish peroxidase (HRP)-conjugated secondary antibody (cat. No. GK500705, GeneTech) at RT for 30–60 m and incubated with 3′-diaminobenzidine (cat. No. GK500705, GeneTech) for 5 m. Subsequently, the slides were counterstained with hematoxylin, dehydrated with a graded series of alcohols, and mounted with coverslips and mounting medium. The staining density was measured using a Leica CCD camera DFC420 connected to a Leica DM IRE2 microscope (Leica Microsystems Imaging Solutions Ltd.). The IHC scores were measured by multiplying staining intensity (0 = no, 1 = weak, 2 = moderate, 3 = strong) with percentage of positive staining (0 = negative, 1 ≤ 10%, 2 = 10–50%, 3 ≥ 50%). UTP11 was defined as low expression when IHC scores were ≤4, and as high expression when IHC scores were >4.