Bx41 microscope

The migration ability of melanoma cells was determined in vitro using Boyden Chambers (Gaithersburg, MD) in which the two chambers were separated with Millipore membranes (8 μM pore size), as detailed previously [15 (link), 24 (link)]. The membranes were examined microscopically and cellular migration was determined by counting the number of stained cells on membranes in at least 3-4 randomly selected fields using an Olympus BX41 microscope with 10x magnification. Representative photomicrographs were obtained using a Qcolor5 digital camera fitted to an Olympus BX41 microscope. Cell migration experiments were repeated to verify the results.

Perfused lungs from infected and uninfected mice were inflated by injecting 1.0 mL of 10% buffered formalin through the same catheter used to perform BALF. Lungs were removed fixed in formalin and embedded in paraffin. Lungs sections of 5-µm thickness were stained with haematoxylin–eosin. Analysis of tissue sections was performed in an Olympus BX41 microscope (Melville, NY, USA) at a magnification of 200×. The number of mononuclear cells in lung tissue was determined by the pointcounting technique across 20 random, non-coincidents microscopic fields in an Olympus BX41 microscope at a magnification of 1000×.

Tissues were isolated and placed in formalin. The tissues were then decalcified, paraffin embedded, and sectioned serially at a depth of 2-4 μ. Paraffin was removed using xylene and tissues rehydrated and immunostained, for instance, using anti-Ucp1 (Novus Biologicals, Centennial, CO, NB100-2828, goat polyclonal, 1:100 dilution) and anti-Mrc1 (Abcam, b64693, rabbit polyclonal, 1:200 dilution) antibodies. Secondary antibodies were linked with Alexafluor 488 and 597 dyes. Tissues were counterstained and covered with ProLong Glass Antifade Mountant with NucBlue (Invitrogen, P36985). Stained tissue sections were examined using an Olympus BX41 microscope (Olympus Corporation of the Americas, Waltham, MA) equipped with a reflected fluorescence system or by confocal microscopy (LSM 510 META, Zeiss, Inc., Thornwood, NY, USA) using a 20X/0.75NA objective lens. To ensure signal specificity, controls were performed and the specific absorption spectrum from each primary-secondary pair was captured. To locate the tissue regions, every 5th slide was stained with hematoxylin (Harris Hematoxylin, American Mastertech, Lodi, CA) and eosin (Eosin Y Phyloxine B solution, Electron Microscopy Sciences, Hatfield, PA). Hematoxylin and eosin images were captured by bright-field microscopy using the Olympus BX41 microscope.

Hematoxylin and eosin (H&E) staining was performed according to standard method. For the paraffin section, liver was fixed in 4% paraformaldehyde (Biosharp, Hefei, China). After 24 h fixation, the tissue was dehydrated and embedded in paraffin. The tissue was then cut into 5 μm slices using a Leica RM22559 microtome (Leica, Wetzlar, Germany). The section was dewaxed, hydrated, and then stained with hematoxylin and eosin. The images were captured using an Olympus BX41 microscope (Olympus Co., Ltd., Tokyo, Japan).
For oil red O staining, cells were fixed with 4% paraformaldehyde at room temperature for 1 h and washed three times with phosphate-buffered solution (PBS, Gibco, New York, NY, USA). Cells were then stained with 0.3% oil red O (Sigma, St. Louis, MO, USA) solution for 1 h at room temperature. After rinsing with PBS, the cells were snapped with an Olympus BX41 microscope (Olympus Co., Ltd., Tokyo, Japan).