*2.4. Immunofluorescence*

Immunofluorescence on placental cryostate sections was done as previously reported [25], with the exception that the incubation with the first antibody was done overnight (ON) at 4 ◦C; the procedure of tissue fixation varied according to the protein to be detected as follows: (1) for E-cadherin, claudins -2 and -7, occludin, and ZO-1, the tissue was fixed in acetone at −20 ◦C for 5 min; (2) for claudins -3 and -4, and JAMs –B and –C, the samples were fixed in ethanol at 4 ◦C for 10 min followed by fixation in acetone at −20 ◦C for 3 min; (3) for ZO-2 and claudins -1 and -5, the tissue was fixed in 4% para-formaldehyde in PBS at RT for 10 min. As primary antibodies, a mouse monoclonal against cytokeratin 18 was employed (Cat. MAB1600, Chemicon International, Temecula, CA, USA; dilution 1:5000), together with one of the following rabbit polyclonal antibodies: anti E-cadherin (Cat. 3195, Cell Signaling Technology, Inc., Danvers, MA, USA; 1:300); anti claudin-1 (Cat. 51–9000, Invitrogen, Camarillo, CA, USA; dilution 1:100); anti claudin-3 (Cat. Ab52231, abcam, Cambridge, MA, USA; dilution 1:100); anti claudin-4 (Cat. 36–4800, Invitrogen, Camarillo, CA, USA; dilution 1:300); anti claudin-5 (Cat. ab15106, Abcam, San Diego, CA, USA; dilution 1:300); anti claudin-7 (Cat. 34–9100, Invitrogen, Camarillo, CA; dilution 1:100); anti occludin (Cat. 71–1500, Invitrogen, Carlsbad, CA, USA; dilution 1:100); anti ZO-1 (Cat. 61–7300, Invitrogen, Carlsbad, CA; dilution 1:300); and anti ZO-2 (Cat. 71–1400, Invitrogen, Carlsbad, CA; dilution 1:100). We also employed mouse monoclonal antibodies anti claudin-2 (Cat. sc-293233, Santa Cruz Biotechnology, Santa Cruz, CA, USA; dilution 1:100); and (1) anti JAM-B (Cat. sc-293496, Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:100); and a rat monoclonal antibody anti JAM-C (Cat. MCA5935, CRAM18F26, SeroTec, Kidlington, UK; dilution 1:100). As secondary antibodies, we employed a donkey antibody against mouse IgG coupled to Alexa594 (Cat. A21203, Invitrogen, Carlsbad, CA; dilution 1:1000), a donkey antibody against rabbit IgG coupled to Alexa-488 (Cat. 21206, Invitrogen, Carlsbad, CA; dilution 1:1000), and donkey antibody against goa<sup>t</sup> IgG coupled to Alexa-488 (Cat. A21208, Invitrogen, Carlsbad, CA; dilution 1:1000).

## *2.5. Relative Mean Fluorescence Intensity Measurements*

Relative mean fluorescence intensity measurements of AJ and TJ proteins at the STB were obtained using ImageJ (ImageJ 1.52n, NIH, Bethesda, MD, USA, 2019) with the Freehand function. An area named A, surrounding the chorionic villi, was selected. Then, another area named B, of the parenchyma of the same villi, immediately below the region of the STB, was selected. The integrated density feature of ImageJ was used to record pixel intensities of each of these two areas. Then, the integrated density of area A minus that of area B was recorded and compared to the fluorescent signal of the STB. Data were derived from three randomly-selected fields per placenta, and the images shown in the figures were one of the quantitated fields.

Data were derived from three images per placenta.
