Axio imager m2 light microscope

FAA-fixed peristomal regions of pitchers were dehydrated through a series of ethanol solutions (50-70-90-96-99.8-99.8% for a minimum of eight hours per step. HistoClear replaced ethanol via a gradual increase in HistoClear (25-50-75-100-100%). HistoClear was subsequently replaced by paraplast at 60 °C (33-50-67-100-100%). Samples embedded in Kendall Paraplast Plus (melting point 56–57 °C) were sectioned at 8 µm with an E. Leitz Wetzlar microtome (Wetzlar, Germany), applied on object-glasses, and stained with Etzolds staining solution (stock: 10 mg Basic Fuchsin, 40 mg safranin, 150 mg Astra Blue, 2 mL Acetic Acid filled up to 100 mL with demi-water) for two hours before being washed with demi-water. Paraplast was removed by washing three times for five minutes with HistoClear. DPX Mountant, a standardized mixture of Distyrene, a plasticizer and xylene, was applied between object-glasses and cover-glasses and left to dry overnight. Microscopy slides were observed with an upright Zeiss Axio Imager, M2 Zeiss light microscope (Jena, Germany). Digital images were obtained with a five-megapixel AxioCam MRc 5 (Jena, Germany) and associated AxioVision SE64 Rel. 4.8 software.

To analyze cytoskeleton changes, hPDLSCs were seeded directly on glass coverslips at a low density and cultured in culture medium containing undiluted extracts of the different endodontic sealers. After 72 h, the cells were fixed with 4% paraformaldehyde in PBS for 30 min and permeabilized with 0.25% Triton-X-100 (Sigma–Aldrich, St. Louis, MO, USA) dissolved in PBS for 20 min. After the cells were blocked with 2% bovine serum albumin, and the actin filaments were stained with CruzFluor594-conjugated phalloidin (Santa Cruz Biotechnology, Dallas, TX, USA), followed by fluorescent-labeled secondary anti-mouse antibody (Invitrogen, Carlsbad, CA) incubation at room temperature for 1 h. DAPI (4, 6 diamidino-2-phenylindole, Sigma-Aldrich, Madrid, Spain) was used to stain cell nuclei (1:1000 dilution). The cells were examined under epifluorescence using an AxioImager M2 Zeiss light microscope (Carl Zeiss, Oberkochen, Germany), equipped with a digital camera, AxioCam MRM (Carl Zeiss).

Cultures were fixed on days one and three with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PB; pH 7.2) for 10 min at room temperature (∼25°C; RT), washed in PB (3x), and treated with 0.5% Triton X-100 in PB for 10 min for cell permeabilization. Cells were incubated with Alexa Fluor™ 488 (green fluorescence)-conjugated phalloidin (1:200, Molecular Probes/Invitrogen, Eugene, OR, USA) for 60 min at RT for actin cytoskeleton labeling. Cells were washed with deionized water and cell nuclei marked with 300 nM 4’,6-diamidino-2-phenylindole, dihydrochloride (DAPI, Molecular Probes, Eugene, OR, USA) for 5 min. The Thermanox^® coverslips were mounted in Prolong Antifade reagent (Molecular Probes, Eugene, OR, USA) and the cells were observed under epifluorescence using an AxioImager M2 Zeiss light microscope (Carl Zeiss Inc., Oberkochen, Germany) outfitted with an AxioCam MRM digital camera (Carl Zeiss Inc., Oberkochen, Germany).

Bacterial strains grown in liquid culture were either directly applied to a microscope slide or previously immobilized on a 2% low-melting agarose in PBS agarose pad and air dried before microscopic analysis. Epifluorescence microscopy was performed using an Axio Imager.M2 light microscope (Carl Zeiss) equipped with Plan-Apochromat 63×/1.40 Oil M27 objective and the AxioCam MR R3 imaging device (Carl Zeiss). GFP, Alexa Fluor 488, eCFP and YFP fluorescence was visualized using filter set 38 (Carl Zeiss; excitation: 470/40 nm band pass (BP) filter; emission: 525/50 nm BP). Chlorophyll auto-fluorescence was recorded using filter set 15 (Carl Zeiss; excitation: 546/12 nm BP; emission: 590 nm long pass). When applicable, cells were previously incubated in the dark at RT for about 5 min with 10 µg ml⁻¹ DAPI in PBS to stain intracellular DNA. For visualization of DAPI fluorescence filter set 49 (Carl Zeiss; excitation: G 365 nm; emission: 455/50 nm) was employed. E. coli BL21 (DE3) cells expressing C-terminally GFP-tagged protein candidates were grown over night in LB and then diluted 1:40 in the same medium the following day. Cells were grown for 2 h at 37 °C, briefly acclimated to 20 °C for 10 min and induced with 0.05 mM IPTG at 20 °C. Protein localization of GFP/YFP-tagged proteins was then observed after indicated time points of cells immobilized on an agarose pad.

For histology, specimens were anaesthetised in chloroform vapour, fixed in alcoholic Bouin’s fluid (Dubosq-Brasil; see [296 ]) for 2 days and washed in 70% ethanol. After dehydration through an ascending ethanol series (70%, 80%, 90%, 95%, 2 × 100%; 2 h each), specimens were incubated for 18 h in methyl benzoate and 24 h in 1-butanol and then embedded in Paraplast Plus® (Sherwood Medical Company, St. Louis, MO, USA) through a series of 1-butanol:Paraplast Plus® mixtures (2:1, 1:2 and then pure Paraplast Plus®) for 24 h each at 60 °C. A complete series of 5-μm sections was generated using a Leitz 1516 microtome (Ernst Leitz, Wetzlar, Germany). The obtained sections were stained with Heidenhain’s Azan staining method ([297 ]; modified by Geidies [298 ]) and analysed under an Axio Imager M2 light microscope (Carl Zeiss Microscopy, Jena, Germany) equipped with an Axiocam 503 colour digital camera (Carl Zeiss Microscopy).

In formalin-fixed, paraffin embedded tissues, the EnVision system (Dako) with anti-Human CD4 (Clone 4B12, Dako), anti-Human CD8 (Clone C8/144B, Dako), anti-Human CD14 (Clone TÜK4, Dako), anti-Human CD56 (Clone 123C3), and anti-Human CD68 (Clone PG-M1, Dako), was used. Briefly, the paraffin sections were deparaffinized and rehydrated in xylene and graded alcohols. After blocking with peroxidase in ChemMate peroxidase-blocking solution (Dako), the slides were incubated with the primary antibodies. After application of the peroxidase-labeled polymer, the slides were incubated with the diaminobenzidine substrate chromogen solution, counterstained with hematoxylin, washed again, dehydrated, and mounted. The immunohistochemical slides were observed using a Zeiss Axio Imager M2 light microscope equipped with a Zeiss Axio Cam HRc Camera to capture images of the placenta. Ten photos were collected per slide, with 40X objective lens. Subsequently, each photograph was analyzed and the number of cells counted for each photo. The number of positive cells was calculated and analyzed using the Image J software (Image J 1.46r Wayne Rasband National Institutes of Health, USA, https://imagej.nih.gov/ij/index.html).