Clearmount

Fluorescent microscopy uptake studies were conducted using FITC-labeled, AS1411-conjugated nanodroplets. Images were acquired using an EVOS FL digital fluorescence microscope (Advanced Microscopy Group, Mill Creek, WA, USA). Human MDA-MB-231 breast cancer cells were plated for 48 hr at a density of 4,000 cells/cm² in glass cell culture dishes (FluoroDish, World Precision Instruments, Sarasota, FL USA). AS1411-conjugated fluorescent nanodroplet emulsions were added to cells at various doses (4%, 2%, 1%, 0.4%, 0.2% and 0% v/v PFP) and incubated for various amounts of time (0, 1, 4, 24, 48, and 72 hr) at 0.4% v/v PFP. Slides were washed with HBSS, fixed with 3.5% paraformaldehyde, stained with 0.05% Hoechst 33342 for 5 minutes at room temperature to detect nuclei, washed twice, and mounted (ClearMount, Invitrogen, Frederick, MD, USA) for at least 3 hours prior to imaging. All images were acquired with identical microscope settings (60% brightness for FITC and 10% brightness for Hoechst). Fluorescence intensity of FITC in cells was quantified using ImageJ.

Isolated whole bladders were pre-fixed in 4% paraformaldehyde overnight and cryoprotected in 30% sucrose. The samples were embedded in OCT compound (Leica Microsystems, Germany), cryosectioned into 10 m thick slices, and mounted on microscope slides. After permeabilization in a solution containing 0.5% triton X-100 and 6% bovine serum albumin for 1 hour, the sections were incubated overnight at 4 °C with primary antibody [Anti- -smooth muscle actin (#ab21027, Abcam), 1:200]. The sections were then washed three times with 1X PBS and incubated with the secondary antibody [Alexa Fluor 594 Donkey Anti-Rabbit IgG (#A21207, Invitrogen), 1:1000] for 1 hour at room temperature. The sections were subsequently washed three times with PBS and stained with DAPI (#R37606, Molecular Probes) to visualize the nuclei. Finally, the sections were rinsed with PBS three times and mounted with Clearmount (#008010, Invitrogen, UK). Confocal fluorescence images were acquired using a ZEISS scanning laser microscope with a 20X or 40X oil immersion objective lens (CLSM780, ZEISS, Germany).

5 μm-thick deparaffinized sections were subjected to heat induced epitope retrieval procedure (HIER) in a citrate buffer (Dako) using a microwave. To eliminate endogenous peroxidase activity, the sections were pretreated at room temperature with 3% H₂O₂ in methanol for 10 minutes. Sections were then incubated with the primary antibody against insulin (Santa Cruz, sc-9168) diluted 1:100 in 1× PBS overnight at +4°C in a moist chamber [26 (link)]. The reaction was detected using Histostain SP kit (Invitrogen) while staining was visualized using AEC chromogen. Slides were counterstained in hematoxylin and mounted using ClearMount (Invitrogen). Each slide was analyzed by microscope (Olympus Provis, Campbell) and photographed (magnification 40×) covering its entire surface. For the determination of beta islet size, number and diameter pancreatic sections stained for insulin and hematoxylin were analyzed. An islet was defined as a cluster of four or more insulin-positive cells. After careful assessment of the entire pancreatic section, five representative islets from five different microscopic fields from five paraffin blocks randomly selected from each group (5 rats from each group) were identified and islet size and diameter were measured. The computed morphometric image analysis and measurements of the digitalized images were carried out using Sform software (Vams).