Alexa fluor 594 conjugated anti mouse immunoglobulin g

Seven days following transplantation, the 10 rabbits were anaesthetized by intraperitoneal injection of 1% pentobarbital sodium at the dose of 50 mg/kg and then sacrificed by an air embolism. The specimens used for pathological examinations were fixed in 10% formaldehyde for 24 h at room temperature, dehydrated using a gradual alcohol series, embedded in paraffin, and sectioned into 30 µm thick sections. Subsequently, the sections were subjected to histological examination with hematoxylin and eosin (H&E), as well as CK3/12 immunochemistry to determine the effect of cell engraftment. Corneas were fixed in formalin, embedded in paraffin and sectioned at 4 µm. For histological staining, slides were refixed, washed and stained with hematoxylin for 5 min and eosin for 3 min at room temperature, respectively. Immunostaining of the corneal sections involved deparaffinization and rehydration in PBS, permeabilization with 1% Triton X-100 for 10 min, blocking with 1% BSA for 1 h, overnight incubation at 4°C with mouse anti-CK3/12 monoclonal antibody (1:100), three 5-min washes with PBS, incubation for 1 h with Alexa fluor-594-conjugated anti-mouse immunoglobulin G (1:400; cat no, A-11032, Invitrogen; Thermo Fisher Scientific, Inc.). and three 5-min washes. Sections were counterstained by incubation with 0.5 µg/ml DAPI for 10 min, followed by immunofluorescence microscopy.

The abdominal aortae of the 23 surviving rats in each group from the pretreatment protocol were harvested and cut using a cryostat (CM1950; Leica) into 8-μm-thick sections. The sections were fixed with 4% paraformaldehyde in phosphate-buffered saline (; pH 7.4) for 10 minutes at room temperature. After rinsing with phosphate-buffered saline, the sections were preincubated with 10% normal goat serum (Nichirei Biosciences, Tokyo, Japan) and incubated overnight at 4°C with mouse anti-CD 68 (Abcam, Cambridge, UK), rabbit anti-MMPs (Abnova, Taipei, Taiwan), rabbit anti-hypoxia-inducible factor (HIF)-1α (Novus Biologicals, Littleton, Colo), and antipimonidazole mouse IgG1 monoclonal antibody (Hypoxyprobe, Inc, Burlington, Mass). Immunoreactivity was visualized using Alexa Fluor 594-conjugated anti-mouse immunoglobulin G and Alexa Fluor 488-conjugated anti-rabbit immunoglobulin G (Molecular Probes, Invitrogen, Carlsbad, Calif). All Alexa-fluoroconjugated secondary antibodies were diluted 200-fold. The slides were mounted in a glycerol-based Vectashield medium (Vector Laboratories, Burlingame, Calif).