Prolong gold antifade mountant without dapi

For serial sections cut at 7 μm in thickness, tissue samples were fixed in 2% PFA overnight at 4°C, dehydrated, and embedded in paraffin. Paraffin slides were first rehydrated to further proceed with antigen retrieval in citrate solution (DAKO, S1699) at 100°C for 20 min. If necessary, then 0.1% hydrogen peroxide was added to methanol to block endogenous peroxidases. The sections were blocked with the appropriate serum (DAKO) and incubated overnight with the following antibodies: rat anti-CD31 (BD Pharmingen, 550274; 1:50), rat anti-F4/80 (Serotec, MCA497F; 1:100), rabbit anti-pimonidazole (Hypoxyprobe, PAb2627AP; 1:100). Appropriate secondary antibodies were used: Alexa Fluor 488–, Alexa Fluor 647–, or Alexa Fluor 568–conjugated secondary antibodies (Molecular Probes; 1:200), biotin-labeled antibodies (Jackson ImmunoResearch; 1:300) and, when necessary, tyramide signal amplification (TSA) system amplification [fluorescein, Cy3, or biotin/3,3′-diaminobenzidine (DAB)] (PerkinElmer, Life Sciences) were performed according to the manufacturer’s instructions. Hoechst solution (Thermo Fisher Scientific, H3570; 1:1000) was used to visualize nuclei. Whenever sections were stained in fluorescence, ProLong Gold Antifade Mountant without DAPI (Invitrogen, P36930) was used. Microscopic analysis was done with an Olympus BX41 microscope and CellSense imaging software.

Eighteen-millimeter glass coverslips were coated with 0.2% gelatin for 1 hour at RT. Subsequently, gelatin-coated coverslips were exposed to 2.5% glutaraldehyde in PBS for 10 min at RT, followed by 70% ethanol for 30 min at RT. After washing with PBS, active aldehyde groups were neutralized with 2 mM glycine in PBS overnight at 37°C. Glycine was removed and the coverslips were washed with PBS before seeding the HUVECs. HUVECs were fixed in 4% PFA for 10 min at 4°C and blocked and permeabilized with blocking buffer [PBS supplemented with 0.3% Triton X-100 and 3% bovine serum albumin (BSA) fraction V] for 1 hour at RT. The following primary antibodies diluted in blocking buffer were incubated overnight at 4°C: goat anti-human TRAIL (R&D, AF375; 1:25), rabbit anti–Ki-67 (Abcam, ab15580; 1:1000) and mouse anti–NF-κB (Cell Signaling Technology, 6956; 1:400). Appropriate secondary antibodies were used: Alexa Fluor 488–, Alexa Fluor 647–, or Alexa Fluor 568–conjugated secondary antibodies (Molecular Probes; 1:200). Hoechst solution (Thermo Fisher Scientific, H3570; 1:1000) was used to visualize nuclei. Coverslips were mounted with ProLong Gold Antifade Mountant without DAPI (Invitrogen, P36930). Microscopic analysis was done with an Olympus BX41 microscope and CellSense imaging software.