Xylol

RPMI-1640 medium, penicillin/streptomycin, and fetal bovine serum (FBS) were purchased from Gibco (Invitrogen, Germany). Hooke kit for EAE induction (myelin oligodendrocyte glycoprotein_35-55 (MOG_35-55) (20 (link)) combined with complete Freund’s adjuvant (CFA) emulsion (21 (link)) and pertussis toxin (PTX):5X) and MOG_35-55 for in vitro stimulation of cells were purchased from Hooke laboratories (EK-0115, Lawrence, MA, USA). Reagents required for histopathological analyses including paraffin, xylol, alcohol, phosphate buffer saline (PBS) and Luxol fast blue were obtained from Sigma (Sigma-Aldrich, Germany). IL-6 ELISA kit and cell proliferation ELISA BrdU kit were obtained from e-Bioscience and Roche, respectively. MS14, an Iranian herbal-marine compound classified as equivalent to food with no observable adverse effect level (NOAEL), was donated by deceased Dr. Ahmadi.

Hematoxylin and eosin (Sigma-Aldrich, St Louis, USA) staining of cell sheets was also performed. Briefly, after detachment and fixation in 4% paraformaldehyde (Sigma-Aldrich, St Louis, USA) for 24 h, samples were paraffin embedded and sectioned in 4 µm thickness. After the sectioning process, the histological samples were placed in a water bath at 50 °C and captured to a histological slide and stored in an oven at 65 °C for 12 h to allow the paraffin remnants to be removed. Sequentially, paraffin removal dehydration protocol was completed using a sequence of xylol (Sigma-Aldrich, St Louis, USA) and alcohol (Sigma-Aldrich, St Louis, USA) solutions and the specimens proceeded a routine H&E staining following the manufacturer's guidelines.

Conceptus samples were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 24 h, dehydrated in dilutions (70%-100%) of ethanol (Sigma-Aldrich), and embedded in paraffin (Tolosa et al., 2003 ). Sections of 5 μm were cut and mounted. Sections were stained using hematoxylin and eosin (Sigma-Aldrich) to investigate blastocyst, surrounding endometrium and embryonic layers formation morphology. Following deparaffinization in xylol (Sigma-Aldrich, Wicklow, Ireland) at room temperature (RT) using two solutions for 10 min each, rehydration in a descending series of etanol concentrations was performed (100%, 2 × 5 min; 5 min each for 90%, 80%, and 70%), followed by distilled water (RT, 5 min). The sections were then stained with hematoxylin for 30 s at RT, washed in running water for 10 min, stained with eosin at RT for 15 s, and washed in running water for 10 min. The slides were then dehydrated in increasing dilutions of ethanol (RT, 5 min 70%, 5 min 80%, 5 min 90%, 2 × 5 min 100%), cleaned in xylene (Sigma-Aldrich) at RT, 2 × 10 min, and mounted on Permount® (SP15-500; Thermo Fisher Scientific, Waltham, MA, USA). The analyses were performed by light microscopy (Nikon Eclipse 80i, Tóquio, Japão).

Slice cultures were fixed in paraformaldehyde (4%, 0.1 M phosphate buffer; Applichem, Darmstadt, Germany) and rinsed in 0.1 M phosphate-buffered salt solution (PBS). Thereafter, slice cultures were exposed for 20 min to toluidine-blue working solution, which was a mixture of 5 ml stock solution (1 g of Toluidine Blue O in 100 ml of 70% ethanol; Sigma-Aldrich) and 45 ml of 1% NaCl solution (pH 2.0–2.5). Thereafter, 96% ethanol (100 ml of 96% ethanol and 4 drops of acetic acid) was used for color-differentiation of the staining. The differentiation step with strong acid removes unspecific staining of weak acidic structures and, thus, increases the contrast between background and stained cells. The process was stopped using 0.1 M PBS, once the differentiation was clearly visible. After brief rinsing with double distilled water, slice cultures were placed on object plates and dried overnight. The slices were then exposed to xylol (Sigma-Aldrich) for 10 min and embedded with Entellan Neu (Merck Millipore, Schwalbach, Germany).