Anti human nuclei antibody

Animal experiments followed institutional guidelines and were approved by Animal Care and Use Committee of Hospital 12 de Octubre with protocol reference PROEX 407/15. Air pouches were generated on the back of 8- to 10-week-old NOD scid gamma (NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ) (NSG^TM) mice by subcutaneous injection of 3 ml sterile air on day 0 and were maintained by reinjecting 2 ml of sterile air on day 3. On day 4, RASF suspensions (2 × 10⁶ cells in 0.5 ml PBS) were injected into the air pouch cavity. At day 9, 15 μg of HIF-1α or control scrambled siRNA duplexes combined with Invivofectamine reagent (Invitrogen) in 200 μl of 5% glucose were injected into the air pouch cavity. Mice (n = 9 per group) were sacrificed on day 11 and the air pouch cavity membrane together with the subcutaneous tissues was dissected and snap-frozen in OCT compound (Sakura, Alphen aan den Rijn, Netherlands).
Frozen sections were fixed in 4% paraformaldehyde and analyzed by immunoperoxidase labelling with anti-human nuclei antibody (Millipore, Temecula, CA, USA). Sections were counterstained with haematoxylin. The whole area of each tissue was photographed and digitalized using a Zeiss Axiocam ERc5s camera and Zen 2012 software (Zeiss, Jena, Germany) on a Zeiss Axio Scope.A1 microscope, and the number of human RASF nuclei per air pouch wall area was quantified.

For immunohistochemistry, excised corneal samples were embedded in frozen section compounds (Surgipath; Leica Microsystems, Nussloch, Germany), and stored at −80 °C until sectioning. Serial sections of 10 µm sections were cut using a HM525 NX cryostat (Thermo Scientific) and collected on polylysin-coated glass slides (Thermo Scientific). Samples were rinsed and blocked in 5% normal goat serum in PBS for 30 min at room temperature. Subsequently, samples were incubated with the primary antibodies at room temperature for 1 hour or at 4 °C overnight. The primary antibodies used were Na⁺/K⁺-ATPase and ZO-1, as well as anti-human nuclei antibodies (Merck Millipore, Massachusetts, USA). Subsequently, samples were labeled with an AlexaFluor 488 conjugated goat anti-mouse IgG secondary antibody (2.5 µg/ml, Life Technology), mounted in Vectashield containing DAPI (Vector Laboratories, California, USA), and visualized using a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

For immunohistochemistry, excised corneal samples were embedded in frozen section compounds (Surgipath; Leica Microsystems, Nussloch, Germany), and stored at −80 °C until sectioning. Serial sections of 8-µm sections were cut using a HM525 NX cryostat (Thermo Scientific, Waltham, MA, USA) and collected on polylysine-coated glass slides (Thermo Scientific, Waltham, MA, USA). Samples were rinsed and blocked in 5% normal goat serum in PBS for 30 min at room temperature (RT). Subsequently, samples were incubated with the primary antibodies at RT for 2 h or at 4 °C overnight. The primary antibody used was antihuman nuclei antibodies (1:50; Merck Millipore, MA, USA). Samples were then labelled with an AlexaFluor 488 conjugated goat anti-mouse IgG secondary antibody (2.5 µg/mL, Life Technology, Waltham, MA, USA), mounted in Vectashield containing DAPI (Vector Laboratories, Burlingame, CA, USA), and visualized using a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany). At least 6 sections per eye were analyzed.