Q500 mc image analysis system

DNMT1 was performed using rabbit anti-DNMT1 antibodies (LifeSpan BioSciences) and a streptavidin-biotin-peroxidase complex for immunohistochemistry staining. Formalin-fixed paraffin-embedded tissue sections were incubated in 3% H₂O₂ in distilled water for 30 min at room temperature, followed by antigen retrieval by boiling the sections in 0.01 M citrate buffer for 20 min for deparaffinization and rehydration. Sections were then washed in 50 mM Tris-HCl (pH 7.6) with 0.05% Tween for 2 min. To block nonspecific binding, all sections were incubated with 5% nonfat dry milk for 30 min at room temperature. The slides were then incubated with anti-DNMT1 antibodies (1:250) for 1 h at room temperature. The reaction was stopped by rinsing the sections with 0.01 M PBS. The sections were then incubated with biotinylated anti-mouse/rabbit IgG serum (secondary antibody), followed by treatment with a peroxidase-labeled streptavidin-biotin complex and diaminobenzidine substrate to visualize the positive cells. Finally, sections were counterstained with hematoxylin, prior to being mounted for examination by light microscopy. DNMT1-positive cells per area in the mouse pancreas were counted using a Q500MC Image Analysis System (Leica, Nussloch, Germany). DNMT1-positive cells were quantified in 20 fields from the cortex and 15 fields from the medulla at a magnification of 200×.

In immunohistochemical/immunocytochemical staining, the positivity was evaluated by image analysis performed using the Leica Q500 MC Image Analysis System (Leica, Cambridge, UK) as previously described [18 (link)]. For picrosirius red staining, a total of 10 fields were randomly chosen per mouse and images were viewed with brightfield illumination as well as with polarization contrast illumination at 40x. Picrosirius red is a birefringent molecule that binds to collagens. The complex fibrillar collagen/sirius red can be detected under polarized light, and the collagen bundles are red, yellow, and green. Collagen expression was quantified under brightfield illumination.

Neutrophil suspensions (1 × 10⁶/mL) were cytospun on culture glass slides positively charged. Then slides were fixed in cold methanol and staining was performed after quenching of endogenous peroxidase with 3% H₂O₂ in methanol. Slides were incubated with primary antibody overnight, followed by incubation with biotinylated antibody for 30 min. Each sample was analyzed for the detection of LRP-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and a labelled polymer HRP anti-mouse from Dako was used as secondary antibody. Staining was completed using the streptavidin-peroxidase method and DAB (3,3′-Diaminobenzidine) (Abcam, Cambridge, UK) [34 (link)]. Cytospins were counterstained with hematoxylin and mounted in Eukitt (Merck Group, Darmstadt, Germany), examined by light microscopy (Leica, Cambridge, UK), and evaluated by image analysis (Leica Q500 MC Image Analysis System, Leica). Isotype-matched IgG monoclonal antibodies (Santa Cruz Biotechnology) was tested as a negative control.

Computerized morphometric analysis of EFs was performed by using the Leica Q500 MC Image Analysis System (Leica Cambridge Ltd., Cambridge, England) on two slides per specimen stained with Verhoeff's iron hematoxylin. All morphometric steps, except the section of the first microscopic field, were automated. EFs were counted electronically and labelled with different colors to facilitate their identification. The software for mathematical morphology calculated the area fraction (Area%) occupied by EF (i.e., pixels of EFs/256”pixels); five fields in each slide were studied.

Fluorescent images were captured by using a Leica DM2000 fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany) coupled to a CCD high resolution cooled camera, using the Leica Application Suite software (Leica Microsystems).
Immunocytochemical staining was evaluated by image analysis as previously described.
²³ (link) For PSR staining, a total of 10 fields per well were randomly chosen and images were viewed with brightfield illumination at 40×. Image analysis was performed using the Leica Q500 MC Image Analysis System (Leica, Cambridge, UK).

A7r5 cells grown on EZ-chamber slides (Merckgroup) and freshly isolated monocytes were treated over night with Mstn (500 ng/ml). Cells were fixed with 2% paraformaldehyde, permeabilized with 0.05%Triton X 100, and stained with 5 U/ml Alexa-Fluor 488-conjugated phalloidin (A-12379 Alexa488 phalloidin; Molecular Probes). Cells were washed three times with PBS and viewed by fluorescence microscopy. Images were captured using the Leica Q500 MC image analysis system (Leica). Single images were digitized for image analysis at 256 gray levels. Imported data were quantitatively analyzed using Q500MC Software-Qwin (Leica). Constant optical threshold and filter combination were used.

To reveal SA β-galactosidase activity, cells were fixed in glutaraldehyde (0.5%) and incubated overnight at 37 °C in a solution containing citric acid (10 mM), potassium ferricyanide (5 mM), NaCl (150 mM), MgCl₂ (2 mM), and 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (1 mg/ml, all Sigma Aldrich)47 (link). Senescence of mNVCM was also revealed by immunostaining using a primary rabbit polyclonal antibody against p16^INK4a (Proteintech), followed by Vectastain secondary antibody, horseradish peroxidase (HRP)-streptavidin, and 3,3′-diaminobenzidine (all from Vector Laboratories) as detection system. Apoptosis was assessed by immunocytochemistry, using a rabbit monoclonal anti-cleaved caspase-3 antibody (clone 5A1E, Cell Signaling Technologies). The percentage of SA β-galactosidase, p16^INK4a and cleaved caspase-3 positive cells was evaluated considering six random fields (20x), taken with the Leica Q500 MC Image Analysis System (Leica).