Blokhen

On day 7 (when differentiated osteoclasts are present), PTHrP-(12-48) (100 nM) in the presence of serum was added to cells for 15, 30, 60, and 90 min. Cells were fixed with 4% paraformaldehyde in PBS at 4°C for 15 min and permeabilized with 0.02% PBS-Triton X-100 for 15 min. Primary antibody block was performed using BlokHen (1:10 dilution in water; Aves Labs, Inc.,Tigard, OR, USA) followed by the addition of primary and secondary antibody using dilutions and incubation times described in Supplemental Table 1.

Slides were incubated in blocking buffer (1X TBST; 3%BSA; 1% normal goat or donkey serum; 0.2% Sodium Azide; 1% Triton X-100), followed by primary antibodies (in blocking buffer) overnight. Secondary antibodies conjugated to Alexa 488 were used to detect the primary antibody. Cells were counterstained with 6-diamidino-2-phenylindole (DAPI) in mounting medium (Vector Laboratories, Inc.) to identify nuclei.
For nestin staining, slides were incubated in BlokHen (Aves Labs, Inc). Chicken anti-nestin was diluted in BlokHen overnight. Fluorescein-labeled goat anti-chicken IgY (1:500 dilution, Aves Labs Cat. #F-1004) was used to detect Nestin. Cells were counterstained with 6-diamidino-2-phenylindole (DAPI) in mounting medium (Vector Laboratories, Inc.) to identify nuclei.

IL-8, PTH1-34 (10 mM), PTHrP(1-37) (1 mM), and PTHrP(12-48) at 1 mM, 100 mM, and 10 mM were resolved on a 12% SDS-PAGE then transferred to a nitrocellulose membrane. The blots were placed in Blokhen (Aves Labs, Inc., Tigard, OR, USA) (diluted 1:10 in water) for 30 min, and washed three times in PBS+ 0.05% Tween-20 before incubating with PTHrP(12-48) antibody and thereafter washed again before incubating with secondary antibody. The primary and secondary antibodies along with antibody dilutions and incubation times are indicated in Supplemental Table 1. Blots were then developed by chemiluminescence using the Bio-Rad Chemidoc MP imaging system. In addition, the blot was stripped for 30 min at room temperature (RT), using Western blot stripping buffer (PBS+ 7ml of β-mercaptoethanol [BME]+ 2% SDS), washed for 30 min and then probed for IL-8 (8 kDa) using a CXCL8/IL-8 specific antibody (Supplemental Table 1) (data not shown).