Axiophot fluorescent microscope

Liver development and regeneration were tested using the transgene Tg(fabp10a:CFP-NTR)⁶⁸ . The embryos used for the analysis were the CFP-positive embryos obtained from a pair of parents with one carrying the heterozygous gene mutation and the other carrying both the heterozygous mutation and an allele of Tg(fabp10a:CFP-NTR). Liver size was measured at 5 dpf. For liver regeneration analysis, the embryos were treated with 10 mM metronidazole for 1.5 days at 3 dpf and analyzed for regeneration at 7 dpf. All analyzed embryos were imaged at a lateral view with head facing right under a Zeiss Axiophot fluorescent microscope, and afterwards genotyped. ImageJ was used to measure the liver areas. Approximately 45 CFP-positive embryos were used for each analysis. Graph shows the mean and s.e.m.

To characterize the immune cell subpopulations, immunofluorescence double staining for LDOC1 and CD68 (Sigma Aldrich, St. Louis, MO, USA), CD56 (Serotec, Oxford, UK) as well as FOXP3 (Abcam, Cambridge, UK), representing markers for leucocyte subpopulations, was performed. The slides were pretreated, comparing the immunohistochemistry procedure. The samples where then incubated for 15 min with the Ultra Vision Protein Block (Thermo Scientific, Lab Vision, Fremont, CA, USA) to prevent unspecific binding of the primary antibody. A mixed solution of the primary antibodies was added at the respective concentrations: LDOC1 and CD56 were diluted 1:100, CD68 1:800 and FOXP3 1:300. The slides were incubated for 16 h at 4 °C in a humid chamber. After washing the slides three times with PBS, they were covered for 30 min in the dark with the fluorophore-labeled secondary antibodies Goat-Anti-Rabbit IgG Cy3 (Dianova, Hamburg, Germany) and Goat-Anti-mouse-AlexaFlour488-IgG (Dianova, Hamburg, Germany). Finally, the slides were rinsed three times with PBS and covered with mounting medium (Vectashield H-1200; Vector Laboratories, Burlingame, CA, USA) containing DAPI for nuclear counterstaining.
Specimens were evaluated under the laser microscope (Axiophot fluorescent microscope; Zeiss, Oberkochen, Germany) using the corresponding software AxioVision (Rel. 4.8, Zeiss, Oberkochen, Germany).

This was carried out on the same day as the flow-cytometry expression analysis. The method used is identical to that in our previously published work [19 (link)] and has been used extensively as an in vitro model for phagocytic receptors [28 ,29 (link)]. In summary, sheep red blood cells (sRBCs) were coated with anti-sheep IgG at room temperature for 1 hour, followed by incubation at 37°C for 20 minutes with C5-depleted serum to form iC3b-opsinized sRBC (sRBC_iC3b). Transfected COS-7 cells were challenged with sRBC_iC3b for 30 minutes at 37°C. External sRBC_iC3b were labeled with an Alexa-488 conjugated anti-IgG antibody and, after fixation and permeabiliation, both internal and external sRBC_iC3b were labeled with an Alexa555-conjugated anti-IgG antibody. COS-7 nuclei were stained with DAPI. A Zeiss Axiophot fluorescent microscope was used visually to analyze the coverslips.

To clearly distinguish between tumor and infiltrating immune cells and to characterize the immune cell subpopulation immunofluorescence double staining for MDR1 and CD45 as common leucocyte marker and other accepted markers for leucocyte subpopulations (CD3, CD56, CD68, CD163, TLR2) was performed. The slides were pre-treated like for immunohistochemistry. To prevent unspecific binding of the primary antibody a blocking solution (UltraVision Protein Block; Thermo Scientific, Lab Vision, Fremont, CA, USA) was applied to the slides for 15 min. The slides were incubated for 16 h with a mixed solution of the primary antibodies (Supplementary file Table S1). After washing the slides two times with PBS, fluorophore-labeled secondary antibodies were applied for 30 min in the dark at room temperature (Supplementary file Table S1). Finally, the slides were covered with mounting medium (Vectashield H-1200; Vector Laboratories, Burlingame, CA, USA) containing DAPI for nuclear counterstaining. All double staining were observed in 20×, 40× and 63× magnification using a confocal laser microscope (Axiophot fluorescent microscope; Zeiss, Oberkochen, Germany) and analyzed with the corresponding software AxioVision. The immune cell subpopulations were quantified by counting positive stained cells for CD68, CD3 and CD56 per field of view (20× lens, n = 12 each).

Cells were processed for immunofluorescence analysis as reported previously^{15 (link)}. Briefly, MDA-MB-231 cells were cultured in 24-well plates containing microscope glass (12 mm) (Thermo Scientific) at 1.8 × 10⁴ cells/well and treated for 24 h with APE 200 μM EqC with and without 1 h pretreatment with 5 μM SP600125 and/or 5 mM NAC. Cells were then incubated for 1 h at 37 °C with a primary antibody to vimentin (1:1000) followed by incubation with secondary antibody conjugated to Alexa Fluor 488 (1:1000) or with primary antibody to E-cadherin and N-cadherin (1:100) followed by incubation with (1:200) FITC-conjugated AffiniPure rabbit anti-mouse and Texas red-conjugated anti-rabbit antibody, respectively. Nuclei were counterstained with DAPI. Vimentin images were performed using Zeiss LSM 700 confocal microscope equipped with a plan apochromat X63 (NA 1.4) oil immersion objective while for E-cadherin and N-cadherin imaging, the coverslips were inverted, mounted in Moviol (Calbiochem, CA) on glass slides, and analyzed using an Axiophot fluorescent microscope (Zeiss). Significant fields were captured and processed using a KS300 system (Zeiss).