Tissue microarrays were constructed from central tissue blocks of third trimester FFPE placentas (n = 100) as described earlier (337 (link)). Briefly, three 20 × 35 mm recipient blocks were made of Paraplast X-Tra tissue embedding media (Fisher Scientific, Pittsburgh, PA, USA). One mm diameter cores from tissue blocks were transferred in triplicate into recipient paraffin blocks using an automated tissue arrayer (Beecher Instruments, Inc., Silver Spring, MD, USA). Five µm sections cut from TMAs were placed on silanized slides and stained with antibodies and reagents (Data S18 in Supplementary Material) either on Ventana Discovery or Leica BOND-MAX autostainers.
Tissue microarray immunostainings were semi-quantitatively scored by three examiners blinded to the clinical information with an immunoreactive score modified from a previously published one (154 (link)). Immunostaining intensity was graded as follows: 0 = negative, 1 = weak, 2 = intermediate, and 3 = strong. All villi in a random field of each of the three cores were evaluated by all examiners, and scores within each core were averaged to represent the target protein quantity of that core. Thus, each placenta had three scores corresponding to the three cores examined.
Tissue microarray immunostainings were semi-quantitatively scored by three examiners blinded to the clinical information with an immunoreactive score modified from a previously published one (154 (link)). Immunostaining intensity was graded as follows: 0 = negative, 1 = weak, 2 = intermediate, and 3 = strong. All villi in a random field of each of the three cores were evaluated by all examiners, and scores within each core were averaged to represent the target protein quantity of that core. Thus, each placenta had three scores corresponding to the three cores examined.
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