Biotin conjugated anti rabbit secondary antibody

Lung tissue was obtained from chronically hypoxic neonatal calves and normoxic age-matched controls. This neonatal calf model of severe hypoxic pulmonary hypertension has been described previously^{53 (link)} and includes the development of PA pressure equal to, or exceeding, systemic pressure as well as remarkable PA remodelling with medial and adventitial thickening, resembling that of human neonatal PH. Indirect immunostaining was performed with rabbit polyclonal anti-OPG antibodies ((1:500), Bioss Antibodies, Woburn, MA, USA) followed by biotin-conjugated anti-rabbit secondary antibody ((1:100), Vector Laboratories) and Streptavidin-Alexa-488 ((1:200), Invitrogen, Carlsbad, CA, USA).

Immunohistochemistry staining of COX-2 was performed as described previously^{18 (link),26 (link)} on formalin-fixed, paraffin-embedded colon segments in sham control and in rats with obstruction and in post-BO state. Sections at 4-μm thickness were blocked with 5% normal goat serum in PBS for 20 min at room temperature, and incubated with the rabbit anti-COX-2 antibody (1:200, Cayman Chemical) and a biotin-conjugated anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA). After incubation with avidin-biotin complex (Vector kit, Vector Laboratories), the sections were stained in diaminobenzidine tetrahydrochloride with 0.03% hydrogen peroxide. As a negative control, sections of the same specimens were processed by the same method but omitting the anti-COX-2 primary antibody.

Immunohistochemistry (IHC) of inflammatory cells was performed on three animals per group. Extracted brains were cut into 3-mm thick coronal slices and immersed in a 20% sucrose solution for 2 hrs at 4°C. Brain slices were embedded in OCT (Sakura Finetek Inc., Torrance, CA, USA) and stored at −80°C. For immunostaining, 10 μm coronal sections were obtained by using a cryostat (Tissue-Tek Cryo3; Sakura Finetek Inc.). Sections were further incubated with 3% H₂O₂ and 10% methanol in PBS, to block endogenous peroxidase. Sections were subsequently incubated overnight at 4°C with primary antibodies against CD68 (ED-1, 1:50 dilution; Abcam) and CD11b (OX42, 1:50 dilution; Abcam), followed by incubation with a biotin-conjugated secondary anti-rabbit antibody (1:200; Vector Laboratories Inc., Burlingame, CA, USA) for 1 hr and streptavidin-conjugated peroxidase (Vecstatin Abc kit, Vector Laboratories Inc.) for 30 min. The slices were developed by incubation (3 min.) with DAB 0.05% (w/v; Dako, Glostrup, Denmark) and visualized under an IX-51 microscope (Olympus Life and Material Science Europe GMBH, Hamburg, Germany), which was attached to a DS-U2 LCD camera (Nikon Instruments Inc., Melville, NY, USA). All slides were evaluated by a blinded, trained observer. Cells were counted in five representative fields of 192 μm² in the infarcted area.