Dfc320 digital camera

Osteoclast numbers were determined according to the American Society for Bone and Mineral Research (ASBMR) system31 in paraffin sections from decalcified tibias stained for tartrate resistant acid phosphatase (TRAP) activity, counterstained with Aniline blue, and imaged using a Leica DMLB2 microscope and DFC320 digital camera.28 For each sample, sections from two separate levels (n = 2 slides) were photographed at magnification ×100 and a 1‐mm × 1‐mm ROI commencing 250 µm below the growth plate was analyzed. Osteoclast numbers per bone perimeter (Oc.N/B.Pm), osteoclast perimeter (Oc.Pm), and osteoclast surface per bone perimeter (Oc.S/B.Pm) were determined in trabecular bone and normalized to total bone perimeter (B.Pm).31 The fraction of bone surfaces that displayed osteoclastic bone resorption was also quantified in high‐resolution BSE‐SEM images.28

Sections from decalcified tibias were stained for tartrate-resistant acid phosphatase and imaged using a Leica DM LB2 microscope and DFC320 digital camera (8 (link), 23 (link)). A montage of nine overlapping fields covering an area of 1 mm² located 0.2 mm below the growth plate was constructed for each bone. BV/TV was measured, and osteoclast numbers and surface were determined in trabecular bone normalized to total bone surface (23 (link)).

After mice were sacrificed on day 56, lungs were fixed with 10% formalin infusion through a tracheal cannula at a constant pressure of 25 cm H₂O. The lungs were immersed in fixative for at least 24 h, after which the left lung was embedded in paraffin. After paraffin embedding, 5 μm sections were cut and de paraffin sections were first deparaffinised. Endogenous peroxidase activity was blocked with 0.3% H₂O₂ (Merck) in methanol for 30 min at room temperature and rehydrated in a graded ethanol series to PBS and paraffin section were stained with hematoxylin/eosin (H&E) for inflammation, periodic acid-schiff (PAS) for goblet cells, Masson’s trichrome for connective tissue, rabbit polyclonal anti-α-smooth muscle actin antibody (Abcam, Cambridge, UK) for smooth muscle cells and rabbit polyclonal anti-Ki76 antibody (Abcam) for proliferating cells according to standard methods. Photomicrographs were taken with an Olympus BX50 microscope equipped with a Leica DFC 320 digital Camera.
Slides were reviewed in blinded fashion by two observers independently and slides were scored on the basis of the percentage of positive stained cells in the following way: -, no positive staining; +/-, less than 25% of cells stained positive; +, 25 to 50% cells stained positive; ++, 50 to 75% cells stained positive.

After lung lavage, lungs were fixated with 10% formalin infusion through the cannula at a constant pressure of 25 cm H₂O. After at least 24 h of fixation lungs were embedded in paraffin. After embedding, 5 μ sections were cut and stained with hematoxylin/eosin (H and E) according to standard methods. Photomicrographs were taken with an Olympus B × 50 microscope equipped with a Leica DFC 320 digital Camera (Akbari et al., 2014 (link); Sagar et al., 2014b (link)).

After euthanasia, wounds with surrounding tissues were excised and fixed with 10% phosphate-buffered (pH = 7.4) formalin. The samples were transversely cut exactly through the center of the wound, dehydrated, and paraffin-embedded according to the routine protocols. Cross sections (5 μm thick) were stained with hematoxylin and eosin (H&E) and Mallory's trichrome stain. For immunostaining, sections were treated with 3% H₂O₂ for 10 min and then with 10% nonimmune goat serum before incubation with rabbit polyclonal antibodies against α-smooth muscle actin (α-SMA) and CD31 (Abcam, UK) or rat monoclonal antibodies against f4/80 (Serotec, UK). Goat anti-rabbit and anti-rat IgG biotinylated antibodies (Vector, USA) were applied and stained with avidin-peroxidase conjugate and diaminobenzidine (Vector, USA). Before CD31 staining, heat-mediated antigen retrieval with Vector Antigen Unmasking Solution (Vector, USA) at 98°C for 40 min was applied additionally.
Samples were analyzed with a DM 5000B microscope equipped with a DFC 320 digital camera (Leica, Germany).

Osteoclast numbers were determined according to the American Society for Bone and Mineral Research system (35 (link)) in paraffin sections stained for TRAP activity using a Sigma TRAP kit (386A-1KT) according to manufacturer's instructions. For each sample sections from 2 separate levels (n = 2 slides) were photographed at ×100 magnification using a Leica DMLB2 microscope and DFC320 digital camera, and a 750 × 750-μm ROI commencing 250 μm below the growth plate was analyzed. Osteoclast numbers per bone perimeter (Oc.N/B.Pm), osteoclast perimeter (Oc.Pm), and osteoclast surface per bone perimeter (Oc.S/B.Pm) were determined in trabecular bone (n = 3–5 mice per genotype, per gender) and normalized to total bone perimeter (B.Pm) (30 (link)). The fraction of the bone surfaces that displayed osteoclastic bone resorption was quantified in high-resolution BSE-SEM images (30 (link)).