Cc mount tissue mounting medium

The expression of Claudin-1 in mouse jejunum was analyzed as per the standard immunohistochemistry protocols with slight modifications [20 (link)]. In brief, paraffin-embedded jejunum sections (5 µm) were deparaffinized using xylene and rehydrated by a gradient of decreasing concentration of ethanol, as per the protocol mentioned in Section 2.10. After rehydration, antigen retrieval was performed in EDTA buffer, pH 8 (10 mM EDTA, 0.05% Tween-20) at 95 °C for 20 min followed by a washing step in 1× PBS, pH 7. The tissue sections were dipped in 3% solution of hydrogen peroxide in methanol to quench the activity of endogenous peroxidase, and after washing with 1× PBS, sections were blocked using 1% BSA (1 h at room temperature) to reduce non-specific binding. Tissues sections were then incubated overnight with goat polyclonal anti-claudin-1 (1:300; sc-22932, Santa Cruz) at 4 °C in a humid chamber. After the overnight incubation, sections were washed (1× PBS, pH 7) and incubated with anti-goat secondary antibody (1:200) for 2 h at room temperature. The color was developed using a standard diaminobenzidine (DAB): hydrogen peroxide reaction for 3 min in the dark to prevent light-induced oxidation of DAB. The sections were mounted with CC/Mount™ tissue mounting medium (Sigma Aldrich, USA) and the images were captured on a light microscope (Zeiss, Oberkochen, Germany).

The ampicillin, IPTG (isopropyl-β-d-1-thiogalactopyranoside), WST-1 reagent, and CC/Mount tissue-mounting medium were obtained from Sigma-Aldrich (St. Louis, MO, USA); the Alamar Blue reagent was from Thermo Fisher Scientific (Waltham, MD, USA); the iRGD peptide was from InvivoChem (Libertyville, IL, USA); sulfo-Cyanine 3 maleimide was from Lumiprobe (Moscow, Russia); pan-caspase inhibitor Z-VAD-FMK was from Santa Cruz Biotechnology (Dallas, TX, United States). All other chemicals were obtained from Applichem (Darmstadt, Germany) unless otherwise specified. All solvents and components of buffer solutions were of analytical grade. Monoclonal antibodies to TRAIL (MAB375) were from R&D systems (Minneapolis, MN, USA); monoclonal antibodies to DR5 (DR5-01-1) and integrin αVβ3 (23C6), secondary antibodies Dylight 488 and mouse IgG1 (15H6) were from GeneTex (Irvine, CA, USA). E. coli SHuffle B T7 cells were from New England Biolabs (Ipswich, MA, USA. Bacterial cells were cultivated using Gibco Bacto yeast extract and Gibco Bacto tryptone (Thermo Fisher Scientific, Waltham, MA, USA). Human glioblastoma U87 and T98G cells were from ATCC (Washington, DC, USA). The cell culture media DMEM, 0.25% Trypsin-Versene solution, and phosphate-buffered saline tablets were from PanEco (Moscow, Russia). Thefetal bovine serum was from HyClone (Cramlington, UK).