Zinc buffered formalin

Experimental dams (infected at E6, E10, or E16) or nonpregnant female mice were euthanized at 3 days after infection. Mice were anesthetized via isoflurane and exsanguination was preformed via cardiac puncture. At the time of euthanasia, the total number of viable and nonviable fetuses was quantified for each pregnant dam. Fetal viability was determined as the percentage of fetuses within uterine horns that were viable. Fetuses were counted as nonviable if they were smaller or discolored compared with gestational age-matched live fetuses or if a fetus was absent at an implantation site (56 (link), 72 (link), 73 (link)). Maternal lungs were collected, separated by lobe, and flash frozen on dry ice for homogenization. The left lung was inflated and fixed in zinc buffered formalin (Thermo Fisher Scientific) for at least 72 hours in preparation for histology. Fetuses and placenta were flash frozen in dry ice or fixed in 4% paraformaldehyde (Thermo Fisher Scientific) for 72 hours at 4°C for IHC. Serum was separated by blood centrifugation at 2,400g for 30 minutes at 4°C. A subset of uninfected pregnant (E16) and nonpregnant adult mice were euthanized and the median liver lobe was collected and flash frozen in dry ice for Western blotting.

Scaffold-only grafts (no NPI) recovered at four weeks were fixed in 10% w/v zinc-buffered formalin (Thermo Fisher, Waltham, MA, USA), embedded in paraffin blocks and subsequently stained using hematoxylin and eosin (H&E) [37 (link),39 (link)]. Similarly, NPI grafts were processed and embedded in paraffin blocks for histological assessment. The presence of insulin positive cells in the grafts was assessed using the avidin–biotin complex (ABC) method with peroxidase and diaminobenzidine as the chromogen in accordance with a previously published method [13 (link)]. The graft total cellular insulin content was extracted in accordance with previously published methods [8 (link),38 (link)] and was analysed using a MesoScale Mouse/Rat Insulin Kit (Meso Scale Diagnostics, Rockville, MD, USA) according to the manufacturer’s protocol.