Lsm 700 inverted confocal microscope

Whole, cell-embedded scaffolds were stained as previously described^{12 (link)}. First, scaffolds were fixated in 4% paraformaldehyde (Electron Microscopy Sciences, USA) for 10 min and subsequently washed with PBS. Next, the cells were permeabilized by adding 0.3% Triton X-100 (Bio Lab Ltd.) for 10 min at room temperature (RT). The scaffolds were then washed with PBS and soaked for 1 h in blocking serum (10% FBS, 0.1% Triton X-100, 1% glycine) at RT. Subsequently, scaffolds were incubated overnight at 4 °C with the following primary antibodies (diluted in blocking solution): mouse anti-human αSMA (1:50, Dako), mouse anti-human collagen IV (1:500, Sigma-Aldrich) and mouse anti-human VE-cadherin (1:150, Santa Cruz). Following four washes of 10 min each in PBS, Cy3-conjugated donkey anti-mouse IgG (1:100, Jackson Immuno-research laboratory, PA) was applied for 3 h. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (1:1000, Sigma-Aldrich). Scaffolds were then washed in PBS and stored in 24-well plates in PBS at 4 °C until observation under a Zeiss LSM700 inverted confocal microscope (Carl Zeiss), with 5× and 10× objective lenses, using ZEN software (Carl Zeiss). Further image analysis was conducted using FIJI (Fiji Is Just ImageJ) software.

Zebrafish embryos were exposed to cigarette smoke or snuff extracts from 48 hpf to 4 days post-fertilization (dpf), 10 embryos per group, anesthetized with 0.5 mg/ml ethyl 2-aminobenzoate (Sigma) and mounted in 1% low melting agarose on a microscope cover slip. The central part of the trunk between the swim bladder and the end of the GI tract were imaged using a LSM700 Zeiss Inverted Confocal microscope with associated software, taking stacks with 5 µm between each slide and 20-30 slides per embryo, covering the entire thickness of the trunk. The presence or absence of a continuous vessel running between the dorsal aorta and posterior cardinal vein (i.e. the thoracic duct) was scored by visual inspection of the resulting micrographs. Clear bulbous fluid-filled spaces around the heart (pericardial edema) or yolk (yolk sac edema) was scored by visual inspection of light micrographs using a Nikon SMZ1500 microscope and NIS-Elements F software.

The Cytrap dual cell cycle phase marker system was used to monitor both the S/G2 and G2/M phases of the cell cycle simultaneously in proliferating cells of embryonic root [17 (link)]. The root tips of control and HC treated Cytrap seeds, placed in a drop of water on a clean slide, were imaged at 40× magnification using a LSM700 Zeiss inverted confocal microscope at excitation wavelengths of 488 nm for the green fluorescent protein (GFP; Figure 4A) and 559 nm for the red fluorescent protein (RFP; Figure 4B), at 0, 2, 5, 8, 10, 16, 18 and 24 h after treatment.

Embryos were fixed with 10% formaldehyde/heptane and devitellinized with Ethanol. Blocking was conducted in BBT for 2 hr at room temperature. A FITC-labeled lectin kit #2 (EY laboratories, San Mateo, CA, USA) was utilized (table below summarizes abbreviations of used lectins). Each lectin was diluted to 1:25 and incubated with fixed embryos overnight at room temperature (RT). Embryos were washed in BBT for 2 hr at RT and Vectashield was added. After overnight incubation at 4°C, embryos were mounted on a slide and imaged with a Zeiss Inverted LSM700 Confocal Microscope using a Plan-Apochromat 63X/1.4 Oil Objective. Macrophages in late Stage 11 embryos were imaged at germband entry and evaluated by eye for enriched staining on macrophages compared to other tissues.

Lectin

Peanut agglutinin

Ulexeuropaeusagglutinin

Wheat germagglutinin

Griffoniasimplicifoliaagglutinin I

Maclurapomifera agglutinin

Griffonia simplicifoliaagglutinin II

Abbreviation	PNA	UEA-I	WGA	GS-I	MPA	GS-II
Lectin	Soybean agglutinin	Dolichos biflorus agglutinin	Concanavalin A	Helix pomatia agglutinin	Limulus poly-phenus agglutinin	Bauhinia purpurea agglutinin
Abbreviation	SBA	DBA	ConA	HPA	LPA	BPA

Cells were grown in glass coverslip bottom Petri dishes (35 mm, No. 0, MatTek, Ashland, MA, USA) at the density of 10⁵ cells. Substances at concentrations of 10 and 100 µL were administered 24 h before observation with an inverted LSM700 confocal microscope (Zeiss, Oberkochen, Germany) equipped with a 20× Fluar (NA = 0.75, ∞, Zeiss, Germany) and a CCD camera (AxioCam HRm, Zeiss, Oberkochen, Germany). Mitochondrial probe MitoTracker^® Orange CMTM/Ros (MTO, 0.2 µM, 15 min, ThermoFisher Scientific, Waltham, MA, USA) was excited by 555 nm CW solid-state laser, and the emission was detected >580 nm. Nuclei of cells were stained with 10 µg/mL Hoechst 33258 (Hoechst, 15 min, ThermoFisher Scientific, Waltham, MA, USA) that was excited with 405 nm laser, and its emission was detected in the range 410–490 nm. Phosphatidylserine was labeled with AnnexinV/FITC (5 µL from the detection Kit, 15 min, Annexin V-FITC Kit, Miltenyi Biotec, Bergisch Gladbach, Germany), which was excited at 488 nm and detected in the range of 500–540 nm. The fluorescence images were analyzed in Zen 2011 software (Zeiss, Oberkochen, Germany) or ImageJ software (National Institutes of Health, Bethesda, MD, USA). The bright-field images were detected with the same microscope.

Embryos were collected on apple juice plates from between 6 and 8.5 hr at 29°C. Embryos were incubated in 50% Chlorox (DanClorix) for 5 min and washed. Embryos were fixed with 17% formaldehyde/heptane for 20 min followed by methanol or ethanol devitellinization except for T antigen analysis, when embryos were fixed in 4% paraformaldehyde/heptane. Fixed embryos were blocked in BBT (0.1M PBS + 0,1% TritonX-100 +0,1% BSA) for 2 hr at RT. Antibodies were used at the following dilutions: α-T antigen (Steentoft et al., 2011 (link)) 1:5, α-GFP (Aves Labs Inc., Tigard, Oregon) 1:500; α-LanA (Kumagai et al., 1997 (link)) (a gift from Stefan Baumgartner) 1:500; α-Vasa (Aruna et al., 2009 (link)) (DSHB, deposited by A. Sprading/D. Williams) 1:25; and incubated overnight at 4°C (GFP) or room temperature (T antigen, LanA). Afterwards, embryos were washed in BBT for 2 hr, incubated with secondary antibodies (Thermo Fisher Scientific, Waltham, Massachusetts, USA) at RT for 2 hr, and washed again for 2 hr. Vectashield (Vector Laboratories, Burlingame, USA) was then added. After overnight incubation in Vectashield at 4°C, embryos were mounted on a slide and imaged with a Zeiss Inverted LSM700 Confocal Microscope using a Plan-Apochromat 20X/0.8 Air Objective or a Plan-Apochromat 63X/1.4 Oil Objective.