Tissue tek o c t optimal cutting temperature compound

The distribution of the elastic liposomes across the buccal tissues was analyzed using CLSM. Briefly, the studies were performed using a vertical static Franz diffusion cell system that was set up as described earlier (Figure 1). The donor compartment was loaded with 500 µL of FITC–insulin solution, SC-EL, or SGDC-EL (equivalent to 200 µg/mL FITC–insulin). The experiments were conducted from the donor to the receptor compartment with constant magnetic stirring (600 rpm) at 37 °C. At predetermined time points (1, 4, and 8 h), the buccal tissues were removed, washed with PBS buffer (pH 7.4), and dried with soft paper tissue. Following this, a Tissue-Tek O.C.T. (optimal cutting temperature) compound (Sakura Finetek USA, Inc., Torrance, CA, USA) was introduced to fix the obtained buccal tissues. Subsequently, the buccal tissues were frozen using liquid nitrogen and were cut vertically using a cryostat (Cryotome FE, Thermo Fisher Scientific, Waltham, MA, USA) into 9-µm-thick slices. These sliced portions were laid onto glass slides, fixed with 4% paraformaldehyde, mounted with coverslips, and analyzed using a Zeiss CLSM 800 with Ariyscan (Carl Zeiss Meditec AG, Jena, Germany).

For paraffin-embedded tissue sections, liver tissues were fixed in 20% formalin, dehydrated in ethanol and xylene, embedded in paraffin wax, and sectioned. After deparaffinization and rehydration of the sections, antigen retrieval was performed by microwaving in 0.01 M citrate buffer (pH 6.0). For frozen tissue sections, liver tissues were directly embedded in Tissue-Tek OCT optimal cutting temperature compound (Sakura Finetek) and sectioned. The frozen tissue sections were initially fixed with 4% paraformaldehyde for 5 minutes and then fixed with methanol for 5 minutes at room temperature. After washing in PBS containing 0.1% Tween-20 and blocking, the sections were incubated with the primary antibodies shown in Supplemental Table 1. After washing, the sections were incubated with Alexa 488-, Alexa 555-, and/or Alexa 647-conjugated secondary antibodies (1:1000; Life Technologies) with DAPI. Stained tissues were viewed using an Olympus IX71 microscope and an Olympus FluoView FV10i confocal laser-scanning microscope.