Serial coronal sections of mouse brains were deparaffinized and rehydrated. After hematoxylin and eosin staining, the histopathological properties were evaluated under a Leica DFC 320 digital camera.
Dfc 320 digital
Manufactured by Leica
The Leica DFC 320 digital camera is a high-performance microscope camera designed for a wide range of applications. It features a CMOS sensor with a resolution of 3.2 megapixels and supports live image preview, image capture, and video recording.
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4 protocols using dfc 320 digital
1
Histopathological Analysis of Mouse Brains
2
Specimen Preparation for SEM and LM Analysis
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For SEM exams, the samples were dehydrated in an ethanol series, critical-point-dried in a Bal-Tec CPD 030 dryer (Balzers, Liechtenstein), mounted on metal stubs, adhered to carbon adhesive tape, and covered with gold in a Bal-Tec SCD 050 sputter coater. Observations and illustrations were performed using a scanning electron microscope (Zeiss EVO-50, Cambridge, UK) at 15 kV.
After surface analysis (SEM), some stages were chosen for evaluation by light microscopy (LM). Samples were dehydrated in an ethanol series and embedded in histological resin [54 ], and transverse and longitudinal 3–3.5 μm thick sections were obtained with a rotary microtome (Leica RM 2245, Wetzlar, Germany). The sections were stained with toluidine blue in phosphate buffer (pH 5.8) [55 (link)] and mounted in water. Observations and illustrations were obtained using a Leica DM 4500 B LM connected to a Leica DFC 320 digital camera. Scales were determined under the same optical conditions.
After surface analysis (SEM), some stages were chosen for evaluation by light microscopy (LM). Samples were dehydrated in an ethanol series and embedded in histological resin [54 ], and transverse and longitudinal 3–3.5 μm thick sections were obtained with a rotary microtome (Leica RM 2245, Wetzlar, Germany). The sections were stained with toluidine blue in phosphate buffer (pH 5.8) [55 (link)] and mounted in water. Observations and illustrations were obtained using a Leica DM 4500 B LM connected to a Leica DFC 320 digital camera. Scales were determined under the same optical conditions.
3
Osteoclast Quantification in Mouse Femurs
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Osteoclast numbers were determined according to the American Society for Bone and Mineral Research system (Dempster et al., 2013) in paraffin sections from decalcified mouse femurs stained for tartrate resistant acid phosphatase (TRAP) activity, counterstained with aniline blue and imaged using a Leica DM LB2 microscope and DFC320 digital camera (Bassett et al., 2014) . A montage of nine overlapping fields covering an area of 1mm 2 located 0.2mm below the growth plate was constructed for each bone. BV/TV was measured, and osteoclast numbers and surface were determined in trabecular bone normalised to total bone surface (Bassett et al., 2014) .
4
Quantifying Ovarian Fibrosis via Trichrome Staining
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Masson's Trichrome stain was performed to determine fibrotic areas in the ovarian tissue by identifying the relative levels of newly formed collagen [18, 19] . Ovaries (six from each group, i.c. control and imatinib treated) were fixed in Bouins' solution and embedded in paraffin. The 4 μm deparaffinized sections were first stained with an acid dye (acid fuchsin), treated with phosphomolybdic acid and finally stained with methyl blue. Old collagen was stained in red, while newly formed collagen fibers were stained in blue. Newly formed collagen fibers were stained in blue. Photomicrographs from the Masson's trichrome-stained sections were taken with an Olympus BX50 microscope equipped with a Leica DFC 320 digital camera (magnification of 100x). As for Picrosirius red staining, fibrotic area calculation was performed automatically with the help of the image processing and analysis software Image J (National Institutes of Health, USA).
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