Digital camera dmx1200

The change in chlorophyll was estimated by the red fluorescence emitted at wavelength 490/525 (excitation/emission) by using Zeiss Axioskop fluorescent microscope equipped with Nikon Digital camera DMX 1200. Chlorophyll amount was quantitatively expressed in pixel intensity by analysing the images using ImageJ. Pixel intensity (indicating the presence of chlorophyll) was measured similarly to the described for H₂O₂ quantification. However, opposite to the readings for H₂O₂, the higher pixel intensity corresponds to higher chlorophyll amount. Zero value of grey corresponds to the lower level of fluorescence and value 255 represents the highest level.

Kidneys collected during necropsy of 24 male Sprague Dawley rats (6 rats per group) were fixed in Bouin’s solution for 24 h, subsequently dehydrated in ascending ethyl alcohol, and then embedded in paraffin. Two serial sections at 4 μm were stained with hematoxylin, eosin and with Masson’s trichrome stain then examined and photographed with a light microscope (Nikon eclipse E600, Melville, NY, USA) associated to a microphotography system (Nikon digital camera DMX1200, Melville, NY, USA). The hematoxylin and eosin stained sections were used to score the severity of glomerular damage, tubular damage, inflammation, and the presence of proteinaceous material in Bowman’s spaces and tubule lumen from 0 to 3 (0 = absent, 1 = mild, 2 = moderate, 3 = intense). The severity of the fibrosis was evaluated on the Masson’s trichrome stained section with ImageJ software (National Institutes of Health) as previously described [27 (link)].

The production of overall ROS was analysed by using the fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCF-DA), according to Sakamoto et al. (2005 ). This dye is non-fluorescent in reduced form and readily permeates the plasma membrane. Once in the cell, non-specific esterases cleave its acetate groups and the dye becomes membrane impermeable, trapped inside the cell. When oxidised by H₂O₂, hydroxyl, peroxyl and other free oxygen radical products, DCF-DA is converted to the green fluorescing form 2′,7′-dichlorofluorescin and ROS appeared in green. Overall ROS were visualised in leaf discs (0.5 cm diameter), collected as described above. The samples were washed with distilled water and incubated in presence of 10 μmol l⁻¹ DCF-DA for 60 min at room temperature, in darkness. The fluorescence emitted from stained ROS was detected under Zeiss Axioskop fluorescent microscope equipped with filter combination excitation/emission wavelength 490/525 nm and with Nikon Digital camera DMX 1200 for imaging.

At slaughterhouse, the brain was immediately removed and divided into two parts by a sagittal paramedian cut. The small part was frozen at −80 °C until further processing while the other part was fixed for 15 days in 10% neutral buffered formalin for histological and immunohistochemical examination. Transversal sections were taken from superior frontal gyrus and hippocampus (dentate gyrus). Precisely, for hippocampal formation we analysed pyramidal cells layer, Cornu ammonis (CA2, CA3 and CA4) fields. Sections were subsequently embedded in paraffin, sectioned at 4 μm and stained with haematoxylin and eosin (HE) and Periodic acid–Schiff (PAS). Histological specimens were examined and photographed with a light microscope (Nikon eclipse E600) associated to a microphotography system (Nikon digital camera DMX1200). Unstained sections from all cases were also evaluated with a fluorescence microscope (AxioSkop2 MOT, Zeiss) associated to a microphotography system (AxioCam MRc5, Zeiss) using blue light excitation (FITC filter; excitation, 455–500 nm; emission, 500–570 nm) in order to detect lipofuscin autofluorescence. The degree of lipofuscin accumulation was estimated as the quantity of neurons containing PAS positive storage material and scored as follows: absent, mild (less than one-third), moderate (between one-third and two-thirds), and high degree (more than two-thirds).

Frozen liver sections (7 μm) were fixed in acetone and subsequently blocked for endogenous peroxidase by incubation with 0.25% of 0.03% H₂O₂ for 5 minutes. Primary antibodies used were against infiltrated macrophages and neutrophils (rat-anti-mouse Mac-1 [M1/70]), and neutrophils (rat-anti-mouse Ly6-C, clone NIMP-R14) (generous gift from Prof Heeringa, Groningen, The Netherlands). 3-Amino-9 ethylcarbazole (AEC) (A85SK-4200.S1; Bio-connect, Huissen, The Netherlands) was applied as color substrate and hematoxylin (4085.9002, Klinipath, Duiven, The Netherlands) was used for nuclear counterstaining. TUNEL staining for apoptosis was performed on frozen liver sections according to the manufacturers' protocol (In situ Cell Death Detection Kit, Roche Applied Science). Sections were enclosed with Faramount aqueous mounting medium (S302580; DAKO, Glostrup, Denmark). For the lipid staining, the neutral lipid marker Oil Red O (ORO; O0625; Sigma-Aldrich) was used.
Paraffin-embedded liver sections (4 μm) were stained with Hematoxylin-Eosin (Eosin, E4382; Sigma-Aldrich). Images were taken with a Nikon digital camera DMX1200 and ACT-1 v2.63 software (Nikon Instruments Europe, Amstelveen, The Netherlands).

Frozen liver sections (7 µm) were fixed in acetone and blocked for endogenous peroxidase by incubation with 0.25% of 0.03% H₂O₂ for 5 minutes. Primary antibodies used were against hepatic macrophages (1:100 rat anti-mouse CD68, clone FA11), infiltrated macrophages and neutrophils (1:500 rat anti-mouse Mac-1 (M1/70)) and neutrophils (1:100 rat anti-mouse NIMP (Ly6G)). 3-Amino-9-ethylcarbazole (Vector laboratories, CA, USA) was applied as color substrate and hematoxylin for nuclear counterstain. Sections were enclosed with Faramount aqueous mounting medium. Additional information concerning the immunostainings is also described elsewhere (29 (link)).
Pictures were taken with a Nikon digital camera DMX1200 and ACT-1 v2.63 software (Nikon Instruments Europe, Amstelveen, The Netherlands). Infiltrated macrophages and neutrophil cells (Mac-1⁺) and neutrophils (NIMP) were counted by two blinded researchers in six microscopical views (original magnification, 200x) and were indicated as number of cells per square millimeter (cells/mm²). Hepatic macrophages (CD68) were counted in six microscopical views (original magnification, 200x) and indicated as the percentage of CD68 positive area (Adobe Photoshop CS2 v.9.0.).

Frozen liver sections (7 µm) were fixed in acetone and blocked for endogenous peroxidase by incubation with 0.25% of 0.03% H₂O₂ for 5 min. Primary antibodies used were against hepatic macrophages (1:100 rat anti-mouse CD68, clone FA11), infiltrated macrophages and neutrophils (1:500 rat anti-mouse Mac-1 (M1/70)), and infiltrated T-cells (1:20, rat anti-mouse CD3). 3-Amino-9-ethylcarbazole was applied as color substrate and hematoxylin for nuclear counterstain. Sections were enclosed with Faramount aqueous mounting medium.
Pictures were taken with a Nikon digital camera DMX1200 and ACT-1 v2.63 software (Nikon Instruments Europe, Amstelveen, The Netherlands). Infiltrated macrophages and neutrophil cells (Mac-1+) and infiltrated T-cells (CD3+) were counted by two blinded researchers in six microscopical views (original magnification, 200×) and were indicated as number of cells per square millimeter (cells/mm²). Hepatic macrophages (CD68) were counted in six microscopical views (original magnification, 200×) and indicated as the percentage of CD68 positive area (Adobe Photoshop CS2 v.9.0., San Jose, CA, USA).