Abc system

Immunohistochemical detection of ANGPTL4 (1:400 dilution; Abcam, Cambridge, MA) and CD34 (1:500 dilution; Covance, Princeton, NJ) was performed in paraffin-embedded tissue sections using premixed biotinylated anti-rabbit, anti-mouse and anti-goat immunoglobulins in phosphate buffered saline (PBS) from an ABC system (Dako, Santa Clara, CA) performed according to the manufacturer’s protocols as previously described[16 (link), 17 (link)]. All immunohistochemical reagents, including antibodies, were identical for all specimens. Images were captured by scanning slides using the Aperio ScanScope program on Aperio Scanscope XT® System (Leica Biosystems, Wetzlar, Germany).

Details for antibodies are provided in table S2. Immunohistochemical (IHC) detection was performed with ABC system (Dako, Santa Clara, CA) according to the manufacturer’s protocol on ischemic retina from patients with PDR (obtained from the Wilmer Eye Institute Ocular Pathology Archives with approval from the Johns Hopkins School of Medicine Internal Review Board) as previously described (66 (link), 67 (link)). Detection of HIF-2α (no. NB-100-122, Novus Biologic) and PAI-1 (SC8979, Santa Cruz Biotechnology) by IHC was performed on cryopreserved human diabetic retina sections using a nitroblue tetrazolium development system using streptavidin alkaline phosphatase. Images were captured by scanning slides with the Aperio ScanScope program on Aperio ScanScope XT System (Leica Biosystems, Wetzlar, Germany).

Streptavidin alkaline phosphatase (APase) immunohistochemistry was performed on cryopreserved tissue sections using a nitroblue tetrazolium (NBT) development system as previously described [21 (link)]. An ABC system (Dako) was performed in paraffin-embedded mouse tissue as previously described [21 (link)]. Primary antibodies used include: HIF-1α (ABCAM), ANGPTL-4 (ABCAM), VEGF (Santa Cruz) after dilution in TBS with 1% bovine serum albumen (BSA). All immunohistochemical reagents, including antibodies, were identical for all specimens.

1 μm thick sections were deparaffinized manually. Endogenous peroxidase activity was blocked with a 3% hydrogen peroxidase–methanol mixture for 20 min at RT. Slides were rinsed in 0.05 mM citrate buffer (pH = 6.0) and incubated at 93 °C for 10 min. The sections were then incubated overnight at 4 °C with the primary anti-CD45 antibody (Abcam ab10558, Cambridge, UK) diluted (1:100) in PBS. After washes, slides were incubated with the biotinylated secondary antibody (DakoCytomation, Glostrup, Denmark) for 15 min at RT. Visualization was performed with the avidin-biotin peroxidase complex method (ABC system, Dako) using aminoethyl carbazole as the chromogen. The extent of interstitial leukocyte infiltration was evaluated quantitatively on total kidney sections using Qupath software version 0.1.2 (Centre for Cancer Research & Cell Biology, Queen’s University, Belfast, UK).

Monoclonal mouse antibody against human Ki-67 (DAKO, Denmark) and human smooth muscle actin (SMA) (DAKO) were applied. The routinely formalin-fixed xenograft tumors were dehydrated in a graded series of ethanol, infiltrated with xylene and embedded into paraffin at a temperature not exceeding 60°C. Three to four micron thick sections were mounted on Superfrost slides (Thermo Shandon, Runcorn, UK) and were manually deparaffinized. To block endogenous peroxidase activity, slides were treated for 5 min at room temperature with 3% H₂O₂ in methanol. Slides were immersed in 0.05 mM citrate buffer (pH=6) and exposed to 750 W microwave for 3×5 min (MFX-800-3 automatic microwave, Meditest, Budapest, Hungary).
Slides were primarily treated with antibody against human Ki-67 (1:40) or SMA (1:100) and incubated for 1 hour at room temperature. After washing with phosphate-buffer-saline, secondary antibody Biotinylated Link (Dako) was applied and incubation occurred for 10 minutes at room temperature. After washing periods for visualization, a standard avidin-biotin peroxidase technique (ABC system, DAKO) was used with diaminobenzidine as chromogen.
The Ki-67-positive tumor cells or SMA-positive vessels per fields of vision were counted manually under light microscope (200-fold magnification), 5 fields of vision per tumor were evaluated.