Dc300f camera

Selected embryogenic structures were evaluated for the transient and stable expression of mGFP 5, 15, and 30 days after bombardment. Somatic embryos were screened for GFP expression using a Leica MZ Fluo III (optic 0.63 Zeiss) fluorescence microscope supplied with a DC 300F camera (Leica Microsystems, Welzlar, Germany) and a plant GFP filter no. 3 from Leica (excitation wavelengths 470–540 nm, emission wavelengths 525–550 nm). Autofluorescence from chlorophyll was blocked using a red interference filter and propidium iodide staining.

Hearts were harvested, fixed in 10% formalin, and embedded in paraffin. IHC was performed as described14 (link). The immunoreactivity was detected using the Novolink polymer detection system according to manufacturer's recommendations (Leica, Nanterre, France). Immunostaining was done using DAB, and tissue sections were counterstained with Mayer’s hematoxylin solution (Merck Millipore, Guyancourt, France). Anti-CTGF antibody, rabbit polyclonal HPA031074 (Sigma Prestige antibodies, Sigma, St Louis, MO) was diluted 1:100 in PBS/ 1% BSA/0.1% Triton. IHC signals were analysed using NIH ImageJ software to quantify the staining within the heart in a 20x field of view under a microscope equipped with a DC 300F camera (digital module R, IM 1000, Leica).

Autophagy was detected by staining autophagic vacuoles with monodansylcadaverine (MDC) as previously described [9 (link)]. HCT116 cells (7 × 10³ cells/well) were plated in 96-well plates and treated for the indicated times. After treatments, cells were stained with 0.05 mM MDC in PBS at 37 °C for 10 min in the dark. Cells were then washed with PBS and analyzed under a Leica DMR fluorescence microscope (Wetzlar, Germany) equipped with a 4′,6-diamidino-2-phenylindole (DAPI) filter system. Pictures were acquired by a computer imaging system (Leica DC300F camera) and three different visual fields were examined for each condition.

To evaluate the formation of autophagic vacuoles monodansylcadaverine (MDC) test was employed as reported [75 (link)]. HCT116 cells (7 × 10³/200 µL culture medium) were plated in 96-wells plates and treated with EtOH. After treatment, cells were incubated with 50 µM MDC for 10 min at 37 °C in the darkness. Then, cells were washed with PBS and analysed by fluorescence microscopy using a Leica DMR (Leica Microsystems, Milan, Italy) microscope equipped with a DAPI filter system (excitation wavelength of 372 nm and emission wavelength of 456 nm). Images were acquired by computer imaging system (Leica DC300F camera, Milan, Italy). Three different visual fields were examined for each condition.

Monodansylcadaverine (MDC) staining was performed to evaluate the formation of autophagic vacuoles as previously reported [41 (link)]. For these evaluations, SK-MEL-28 cells were plated in 96-well plates and treated with ITF2357. After 16 h treatment, cells were incubated with 50 µM MDC (Sigma Aldrich, Milan, Italy) for 10 min at 37 °C in the darkness. Cells were then washed with PBS and analyzed by fluorescence microscopy using a Leica DMR (Leica Microsystems, Milan, Italy) microscope equipped with a DAPI filter system (excitation wavelength of 372 nm and emission wavelength of 456 nm). Images were acquired by computer imaging system (Leica DC300F camera, Milan, Italy). Three different visual fields were examined for each condition.

The mean intensity of TH (+) branches was measured in the injured and control sides in six anatomic levels along the substantia nigra pars reticulata (SNpr) and the striatum per rat. Background intensity was excluded from the immunohistochemically stained area. TH (+) neurons were counted in 8 slices of the SNpc (2 caudal, 4 medial and 2 rostral) per rat, as described previously [31 (link), 68 (link)]. The total number of rats was three per every time point for the BSSG group, and six for the UT and mock groups; the rats of the latter group belonged to days 15 and 120 after the DMSO injection since there was no statistically significant difference when compared with the UT group. The immunohistochemical staining was analyzed with a Leica DMIRE2 microscope using the objectives 20x (SNpc) and 5x (striatum). Images were digitized with a DC300F camera (Leica; Nussloch, Germany). ImageJ software v.1.46r was used to measure the total area density of α-synuclein aggregates and TH (+) fibers in the SNpc, and optical density in the striatum. Fiji, an Image J complement, was used for color decomposition from the double staining of β-Gal staining with TH, GFAP, or Iba1 immunohistochemistry.

Microscopic analyses were performed using epifluorescence DM2000 LED microscope (Leica) equipped with a DF450 C camera (Leica) or an Eclipse TE2000-S inverted microscope (Nikon) equipped with a DC 300F camera (Leica). Images were acquired using the Leica Application Suite 4.2 software (Leica).