Iris digital imaging system

Lumbar DRG, spinal cord, and glabrous skin biopsies were prepared from mice at 6 weeks. Sections (14 μm for DRG and 30 μm for spinal cord and glabrous skin) prepared using a Leica CM3050s cryotome (Leica Microsystems, Wetzlar, Germany) were permeabilized with 0.2% Triton-X100 for 1 h. For cultured DRG neurons, cultures were fixed in 4% paraformaldehyde for 15 min, followed by permeabilization with 0.1% Triton-X100 for 10 min and blockade with 3% bovine serum albumin in phosphate-buffered saline. Samples were then stained with anti-GPR171 (rabbit, 1:200; Abcam) anti-TRPV1 (mouse, 1:100; Abcam), anti-TRPA1 (sheep, 1:200; Antibodies-online, Aachen, Germany), anti-TRPV4 (sheep, 1:500; Invitrogen, Rockford, IL. USA), anti-CGRP (goat, 1:2000; Abcam), anti-NF200 (mouse, 1:100; Sigma-Aldrich), anti-NeuN (mouse, 1:100; Chemicon, Temecula, CA, USA), and/or anti-PGP9.5 (mouse, 1:50; Abcam) antibodies. After 24 h at 4 °C, sections were stained with secondary antibodies (Alexa 594-labeled or Alexa 488-labeled secondary antibodies; Life Technologies) for 1 h at room temperatures and placed under a coverslip, followed by image acquisition with an iRiS Digital imaging system (Logos Biosystems, Anyang, Korea) or Zeiss LSM 800 confocal microscope (Carl Zeiss, Oberkochen, Germany).

The intracellular drug release and cellular uptake from G4.5-DOX and Na-DOC-hyd-RESV were qualitatively assessed by comparing the intensity of the red (DOX) and green (RESV) fluorescence emitted by the drugs incorporated by HeLa cells with that of the free drugs (control) (using iRiS™ Digital Imaging System from Logos Biosystems, Annandale, VA, USA). The HeLa cells were seeded in DMEM supplemented with 1% (w/v) penicillin, 10% (w/v) FBS and incubated at 37 °C in a humidified atmosphere of 5% CO2. The HeLa cells were grown to a density of 1 × 10⁵ mL⁻¹ on a coverslip in a 33 mm confocal dish for 24 h. Then again, the cells were co-incubated with free drugs (DOX or RESV, or RESV+DOX) and G4.5-DOX or Na-DOC-RESV at equivalent DOX and RESV concentration of 2.5 μg mL⁻¹ and 7.5 μg mL⁻¹, respectively. The cells were incubated for about 2, 4 and 8 h with G4.5-DOX NPs and 4, 8 and 12 h with Na-DOC-hyd-RESV. Subsequently, the dishes were emptied, washed thrice with 0.1 M PBS and stained with 500 µL 4′,6-diamidino-2-phenylindole (DAPI) (0.3 μM) for 15 min, followed by fixing with 4% (w/v) paraformaldehyde for another 20 min.

Lumbar DRGs from mice were fixed in 4% paraformaldehyde for 4 h, and cryoprotected overnight at 4°C in 30% sucrose. Then 14-μm sections were prepared using a Leica CM3050s cryotome (Leica Microsystems, Wetzlar, Germany) and were permeabilized with 0.2% Triton-X100 for 1 h and then stained with anti-GnRHR (rabbit, 1:1000; Abcam), anti-GnRH (rabbit, 1:1000, Invitrogen), anti-cGRP (goat, 1:2000; Abcam), and anti-NF200 (mouse, 1:100; Sigma-Aldrich) antibodies. After 24 h at 4°C, sections were stained with secondary antibodies (Alexa 594-labeled or Alexa 488-labeled secondary antibodies, and Alexa 594-conjugated IB4, Life Technologies) for 1 h at room temperatures and placed under a coverslip, followed by image acquisition with an iRiS Digital imaging system (Logos Biosystems, Anyang, Korea) and/or Zeiss LSM 800 confocal microscope (Carl Zeiss, Oberkochen, Germany).