Ab201340

Two control and two DLAAA animals were euthanized and their eyes enucleated for histopathology. After removing the excess orbital tissue, eyes were placed in Davidson’s Fixative for 24 hours followed by 10% neutral buffered formalin. One fixed eye from each animal was processed, embedded in paraffin, sectioned and stained by HSRL (Mount Jackson, VA) for histopathology, immunofluorescence (IF) and immunohistochemistry (IHC). Histopathology was performed on sections using standard Hemotoxylin and Eosin staining. IF was performed on additional sections of eyes using anti-glial fibrillary acidic protein (GFAP) primary antibody (Cell Signaling - 3670s, 1:500, 1 hour at room temperature), followed by incubation in AlexaFluor 488 secondary antibody (Invitrogen – A-11001, 1:1000, 30 mins at room temperature). IHC was also performed using the following primary antibodies: anti-von Willebrand factor (vWF) (Thermo Scientific - MA514029, 1:200, 1 hour at RT), anti-CD68 (Abcam – ab201340, 1:200, 1 hour at RT), anti-CD105/endoglin (Abcam – ab219362, 1:1700, 1 hour at RT), anti-VEGF (Novus – NB100–664, 1:1000 at RT). This was followed by incubation in either Mouse on Farma HRP (Biocare Medical, BRR4002) or Rabbit on Farma HRP (Biocare Medical, BRR4009) and visualization using Betazoid DAB (Biocare Medical, BDB2004).

On days 7, 14, and 21 after the operation, the wound and adjacent skin tissues were collected, fixed with 4% paraformaldehyde and embedded in paraffin. The embedded samples were sectioned (5 μm thickness) and stained with hematoxylin-eosin (H&E), Masson’s trichrome and Picrosirius Red (Sigma-Aldrich, USA). H&E staining was used to evaluate granulation tissue formation and re-epithelialization. Granulation tissue and newborn epidermis were calculated with ImageJ software. Masson’s trichrome and Picrosirius Red staining were used to analyze collagen deposition and collagen type I/III ratios. In order to evaluate angiogenesis at days 7 and 21 after surgery, CD31 (1:200, Abcam, USA) antibody, α-SMA (1:200, Abcam, USA) antibody and VEGF (1:200, Abcam, USA) were used for immunofluorescence (IF) staining and immunohistochemical (IHC) staining. To evaluate inflammation at days 7 and 21 post-operation, the Pan macrophage marker CD68 (ab201340, Abcam) antibody and M2 macrophage marker CD206 (Ab64693, Abcam) antibody were used for immunofluorescence staining. The nucleus was stained by DAPI. Immunofluorescence and immunohistochemistry stained images were obtained with a confocal microscope (Olympus FV10 inverted microscope, Japan) and an inverted microscope (Axio Vert A1, Carl Zeiss, Germany), respectively. Image J software was also used to analyze the results.