Unicel dxi 600 access immunoassay system

Plasma samples obtained from peripheral blood were stored in aliquots (−80 °C) until vitamin D levels were measured in a single batch in October 2017. Vitamin D levels were measured using the UniCel DxI 600 Access Immunoassay System (Beckman Coulter, CA, USA) through the Access 25(OH) Vitamin D Total Assay which detects both 25(OH)vitamin D2 and 25(OH)vitamin D3. For this study, we used the 25(OH)D as an indicator of vitamin D levels in plasma. The analysis was performed at Laboratorios Ramírez, a CLIA-certified laboratory located in Ponce, Puerto Rico.

Pigs were placed into a hammock sling for blood collection from the radial vein. Prior to blood collection, technicians applied lidocaine cream (EMLA Topical Cream, Actavis, Parsippany, NJ, USA) to the two front legs around the location of radial vein collection. Technicians used ~½ tsp and rubbed it onto the pigs' legs. The lidocaine was applied at least 5 min prior to blood collection, and not longer than 20 min. Technicians collected ~2 mL of blood into barrier-free, additive-free silica/PET serum vacutainer collection tubes. The clotted blood was centrifuged at 1,300 × rcf for 10 min under ambient conditions to separate the serum. Serum samples were harvested and transferred into microtubes and subsequently stored frozen at −60°C until thawed for determination of cortisol level by automated chemiluminescent immunoassay using the Unicel DxI 600 Access Immunoassay System (Beckman Coulter, Inc., Brea, CA, USA).

The scaffolds were put on glass holders and fixed with an alcoholic fixation solution to prepare them for Ki-67 immunostaining. After rehydration, antigen retrieval was done with heat at pH 6 with target retrieval solution (Dako, Germany, Waldenbronn) according to the manufacturer’s instructions. The immunostaining was conducted on an automated immunohistochemistry staining system (intelli Path FLX, Biocare, USA, Ca, Pacheco) with a mouse anti Ki-67 monoclonal antibody (Zytomed, mouse anti Ki-67 (MSK018) Dako, Germany, Waldenbronn) diluted 1:200 and incubated for 30 minutes at room temperature. For detection the Zytochem-plus HRP kit was used (Zytomed, Germany, Berlin) with incubation times of 20 minutes each from the biotinylated secondary antibody and the Streptavidin conjugate and stained for 12 minutes with a DAB substrate (DAB Substrate Kit, Zytomed, Germany, Berlin) and counterstained with haematoxylin (Dako, Germany, Waldenbronn).
Estradiol and Progesterone concentrations during follicle cultivation with PCL and PCL/gel were evaluated by analyzing removed culture medium during replacement; supernatants from day 2 and day 10 were analyzed in duplicates by using the Access Estradiol and Progesterone Reagent Packs (Beckman Coulter, USA, Ca Brea) with the UniCel DxI 600 Access Immunoassay System (Beckman Coulter, USA, Ca Brea), according to the manufacturer.