3 amino 9 ethylcarbazole kit

Immunohistochemical analysis was performed on cryosections taken from the center of each wound [2 (link),7 (link),33 (link)]. Sections were air-dried, fixed in cold acetone, washed with PBS, quenched with 0.3% hydrogen peroxide, and washed with PBS. Sections were blocked with buffer containing 3% bovine serum albumin and then incubated with F4/80 antibody to label macrophages (1:100, eBioscience, San Diego, CA, USA) or Ly6G antibody to label neutrophils (1:100, BD Pharmingen, San Diego, CA, USA). Sections were then washed with PBS and incubated with biotinylated anti-rat secondary antibody (1:200, Vector Laboratories, Burlingame, CA, USA). After a wash with PBS, sections were incubated with avidin D-horseradish peroxidase (1:1000) and developed with a 3-amino-9-ethylcarbazole kit (Vector Laboratories). Digital images were obtained using a Nikon Instruments Eclipse 80i microscope with a 20x/0.75 objective, a DS-Fi1 digital camera, and NIS Elements software. The percent area stained in each image was then quantified as described above for trichrome and CD31.

Immunohistochemical analysis was performed on cryosections. Sections were air-dried, fixed in cold acetone, washed with PBS, quenched with 0.3% hydrogen peroxide, and washed with PBS. Sections were blocked with buffer containing 3% bovine serum albumin and then incubated with F4/80 antibody to label macrophages (1∶100, eBioscience, San Diego, CA) or Ly6G antibody to label neutrophils (1∶100, BD Pharmingen, San Diego, CA). Sections were then washed with PBS and incubated with biotinylated anti-rat secondary antibody (1∶200, Vector Laboratories, Burlingame, CA). After a wash with PBS, sections were incubated with avidin D-horseradish peroxidase (1∶1000) and developed with a 3-amino-9-ethylcarbazole kit (Vector Laboratories). Digital images were obtained using a Nikon Instruments Eclipse 80i microscope with a 20×/0.75 objective, a DS-Fi1 digital camera, and NIS Elements software and the percent area stained in each image was then quantified as described above for trichrome and CD31.