Prolong gold mountant with dapi

RNAScope is a patented RNA in situ hybridization technology developed by ACD, designed for detecting RNA in fixed tissues. We prepared colon fragments by fixing them in 10% NBF for 16 hours, then slicing them into 5-µm sections. Following deparaffinization and rehydration, we adhered to the vendor’s manual for in situ hybridization (FISH). The procedure entailed the following steps (1 (link)): Pre-treatment with H₂O₂, target retrieval, and proteinase plus, among other steps (2 (link)). Hybridization of the 16s rRNA probe to the RNA target using a hybridization oven for 2 hours at 40°C (3 (link)). Signal amplification through a series of steps, including Amp1, Amp2 and Amp3. Subsequently, we visualized the signal using TSA-fluorescent (TSA-cyanine 3, PerkinElmer LIFE SCIENCES) detection methods. Samples were counterstained with ProLong™ gold Mountant with DAPI (Invitrogen, cat no. p36931) for further analysis. The fluorescent images were captured with a DMC2900 Color Camera using the Leica Thunder Microscope Imager System, manufactured in Wetzlar, Germany. These images were then processed and assembled with Adobe Photoshop. In the final analysis, we focused on attributes like bacterial load, bacterial translocation, and the morphology of the epithelium.

For intracellular trafficking analysis of the dendritic conjugates, U-87 MG cells were plated on 13 mm cover glass (1 × 10⁵ cells/cover glass) and were allowed to form a monolayer for 24 hr. Cells were then added with dye-labeled dPGS-PTX-IDCC or dPG-PTX-IDCC (0.5 µM equivalent PTX). Five minutes prior to each time point, cells were incubated with LysoTracker Red DND-99 (Thermo Fisher Scientific, MA, USA) and then washed and fixed using 4% paraformaldehyde (PFA) for 20 min. Cells were then stained with anti α-tubulin antibody (BioLegend, CA, USA) for 2 hr, followed by incubation with FITC-labeled secondary antibody (Jackson ImmunoResearch Laboratories Inc., PA, USA) for 1 hr. Cover glasses were then mounted by ProLong Gold mountant with DAPI (Thermo Fischer Scientific, MA, USA). Cellular uptake and co-localization between the polymer and lysosome were monitored with a Leica TCS SP5 confocal imaging system (Leica Microsystems, Wetzlar, Germany).
For in vivo intracranial tumor targeting analysis of the dendritic conjugates, 5 µm thick brain sections were mounted with ProLong Gold antifade mountant with DAPI (Thermo Fischer Scientific). Fluorescent signals of mCherry-labeled tumor cells, IDCC-labeled dendritic conjugates and DAPI were imaged using Leica TCS SP8 confocal imaging system (Leica Microsystems, Wetzlar, Germany).