Fish skin gelatin

IFA was performed to ascertain and detect intracellular development of microsporidia in relation to staurosporine induction of apoptosis in individual THP-1 cells. The TMR-red-stained cells were blocked in 0.2 % v/v fish skin gelatin (FSG; Sigma-Aldrich) in TBS for 30 min and then incubated in 10% normal goat serum (NGS; GIBCO) for 40–60 min at room temp. A rabbit polyclonal antiserum raised against a combination of microsporidia species (E. cuniculi, E. intestinalis, E. hellem and V. corneae) was diluted 1:200 in 10% normal goat serum (NGS), centrifuged, and applied to experimental wells for incubation for 60 min at room temperature or overnight at 4 °C. The slides were washed sequentially in TBS containing 0.2% cold water fish skin gelatin (FSG; Sigma-Aldrich) and then TBS containing 0.1% Triton X-100. The cells on slides were then incubated with Alexa 488 (Molecular Probes, Eugene, OR)-conjugated goat-anti-rabbit IgG diluted 1:1000 in 10% NGS for 60 min at room temp. Slides were then rinsed with TBS and counterstained with To-Pro-3 (far red) fluorescent dye (Molecular Probes) to identify host cell nuclei. Slides were examined by inverted confocal laser scanning microscope Leica TCP SP2. Approximately 12–14 optical sections at intervals of 0.3–0.5 µm were recorded for the presence of microsporidia.

Confocal microcopy was preformed as described (Chang et al., 2011 (link); Lin et al., 2015 (link)). Using transmitted light morphology, tubulin bridge staining, and DNA staining, specific identification of telophase or cytokinetic sibling pairs with exclusion of any spurious neighboring-but-unrelated cell pairs is achieved (Lin et al., 2015 (link)). For fixed cell imaging, cells were adhered to poly-l-lysine (Sigma) coated coverslips (#1.5 Thermo Fisher) and treated with freshly prepared 3.7 % PFA (Sigma) diluted in cell culture media (pH 6.8) in order to preserve mitochondrial morphology and MitoTracker Red FM fluorescence intensity. Following brief treatment with 0.1% triton cells were stained with antibody against α-Tubulin (clone YOL1/34 Abcam; 1:300) and then anti-rabbit antibody conjugated to AlexaFluor568 (Thermo Fisher) in a PBS solution containing 0.01 % saponin and 0.25 % fish skin gelatin (Sigma). For live cell imaging, cells were transferred to poly-l-lysine coated 8-chambered coverglass wells (#1 LabTek) in warm cell culture media and allowed to adhere briefly before imaging. Images were acquired on inverted confocal microscopes (Zeiss LSM710 or Nikon Ti Eclipse) and processed using ImageJ (v.1.46r) or Fiji (v.2.0) software. Total fluorescence in defined regions of single cells was quantitated using the integrated density function in ImageJ.

Placenta samples were fixed in neutral-buffered formalin for 3 days, processed, embedded in paraffin, and sectioned (5 μM-thick) for immunohistochemistry. Sections were blocked with 2% goat serum and 0.2% fish skin gelatin (Sigma) in 1X PBS for an hour at room temperature, then incubated with primary antibodies (rabbit anti-MTA3 antibody, mouse anti-hCG antibody (clone 5H4-E2), mouse anti- HLAG antibody (clone 4H84), all from Abcam; mouse anti-Ki67 antibody from Dako; or nonspecific mouse or rabbit IgG as negative controls) at 4 °C overnight. After three washes with 1X PBS with 0.1% Tween-20, sections were incubated with secondary antibodies (Alexa Fluor 488- or 568-conjugated goat anti-mouse or anti-rabbit, from Invitrogen) and DAPI for one hour at room temperature, then mounted with anti-fade mounting solution (Invitrogen) and examined using a Leica fluorescence microscope. Adjacent sections were also stained using hematoxylin and eosin, according to standard protocols, and examined using an Olympus light microscope.