Pimonidazole

60 mg/kg bodyweight pimonidazole hydrochloride (Hypoxyprobe-1™ kit, Chemicon (Millipore), Livingston UK) diluted in sterile phosphate buffered saline (PBS, Invitrogen Ltd. Paisley UK) was administered by intraperitoneal injection 3 hours prior to euthanasia as previously described [26] (link). Animals were terminally anaesthetised and eyes were fixed by intracardiac perfusion with 1% paraformaldehyde in PBS pH 7.4 and post fixed in 1-4% paraformaldehyde at room temperature. Retinal cryosections were incubated for 1 hour at room temperature in PBS with 1% bovine serum albumin (BSA, Sigma Aldrich Ltd. Gillingham, UK) and 5% normal goat serum (Abd serotec, Kidlington UK) and for retinal flatmounts, 0.05% (v/v) Triton X-100 (Sigma Aldrich Ltd., Gillingham UK) was added to the blocking solution. For retinal cryosection analysis (n = 3 eyes for each time-point), 12 µm retinal sections were incubated with anti-pimonidazole antibody (FITC conjugated mouse anti pimonidazole in Hypoxyprobe-1™ kit, Chemicon, Millipore Ltd. Livingston UK) at 1∶50 dilution in blocking solution for 1 hour at room temperature. For retinal flatmount examination (n = 3 eyes for each time-point), eyecups with the cornea and lens removed were incubated with anti-pimonidazole antibody at a dilution of 1∶100 in blocking solution for 4 hours at 37°C. Counterstaining with TRITC conjugated Bandeiraea simplicifolia (BS) lectin (Sigma Aldrich, Gillingham, UK) was performed at a concentration of 0.1 mg/ml in PBS overnight at 4°C. Retinas were washed extensively in PBS and dissected from the eyecup, radial cuts were made to flatten the retina, and the retinas were mounted with fluorescent aqueous mounting medium (Dako Ltd., Ely UK) ganglion cell layer uppermost using a coverslip.

BALB/c mice (female, 8-week-old, purchased from CLEA Japan) were prepared. C26 cells (ca. 1 × 10⁶ cells) were suspended in serum-free RPMI-1640 and subcutaneously inoculated to both hindlegs of the mice. After 10 days, Pimo-yne and pimonidazole hydrochloride were dissolved in the vehicle (5% v/v DMSO, 45% v/v polyethylene glycol (PEG)-400, 50% v/v DPBS(–)) and intravenously injected into the mice at 30 mg/kg each. After 2 h, the mice were anesthetized by isoflurane and perfused by sterile heparinized DPBS(–) and 4% PFA in 0.1 M PB. The tumors were extracted and fixed in 4% PFA in 0.1 M PB at 4 °C overnight. After DPBS(–) wash, the tumor tissues were immersed in sucrose solution, embedded in optimal cutting temperature (OCT) compound (#4583, Sakura Finetek Japan), and frozen on dry ice. The samples used in the following experiment were randomly chosen from the two tumors obtained from each leg of the mice. The tissue sections at a 10 μm thickness were prepared with a cryostat (CM1950, Leica) and permeabilized with 0.2% w/v Triton X-100 in DPBS(–) for 10 min. After DPBS(–) wash, the sections were treated with a CuAAC reaction solution (25 μM Cy5-PEG₄-azide or CF647 azide, 1 mM CuSO₄, 2 mM BTTAA, 5 mM NaAsc, 10% v/v DMSO in DPBS(–)) in the dark at r.t. for 1 h and washed with 0.2% w/v Triton X-100 in DPBS(–) three times. Then, the sections were washed with DPBS(–) three times and blocked with Protein Block Serum-Free in the dark at r.t. for 30 min. After the blocking solution was removed, the sections were incubated with anti-pimonidazole rabbit polyclonal antibody (1:100 in blocking solution, #Pab2627, Hypoxyprobe) in the dark at 4 °C overnight. (NOTE: Pimo-yne was not detected by the anti-pimonidazole antibody (data not shown)). The sections were washed with DPBS-T (0.05% v/v Tween-20 in DPBS(–)) three times and incubated with Alexa Fluor 488 (AF488)-conjugated anti-rabbit antibody (1:100 in blocking solution, #4412S, Cell Signaling Technology). The sections were washed with DPBS-T three times and mounted with DAPI fluoromount-G (#0100-20, SouthernBiotech). The sections were imaged by a fluorescence microscope (BZ-X800, Keyence) with Plan Apochromat 4X (BZ-PA04) or Plan Apochromat 10X (BZ-PA10). The acquired images were processed using Fiji [24 (link)].