Osteopontin Recombinant Rabbit Monoclonal Antibody [PSH14-11]

Immunohistochemical analysis of paraffin-embedded mouse knee joint tissue with Rabbit anti-Osteopontin antibody (HA723601) at 1/9,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA723601) at 1/9,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Application: IHC-Fr

Species: Mouse

Site: kidney

Sample: Frozen section

Antibody concentration: 1/500

Antigen retrieval: Not required

Application: IHC-Fr

Species: Rat

Site: kidney

Sample: Frozen section

Antibody concentration: 1/500

Antigen retrieval: Not required

Western blot analysis of Osteopontin on different lysates with Rabbit anti-Osteopontin antibody (HA723601) at 1/5,000 dilution.

Lane 1: RAW264.7 cell lysate
Lane 2: RAW264.7 treated with 100ng/mL LPS for 4 hours, add 1μg/mL BFA for last 3 hours cell lysate
Lane 3: RAW264.7 cell lysate
Lane 4: RAW264.7 add 100nM TPA overnight, then treated with 100ng/mL LPS for 4 hours, add 1μg/mL BFA for last 3 hours cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 32 kDa
Observed band size: 60 kDa

Exposure time: Lane 1-2: 46 seconds; Lane 3-4: 4 seconds; ECL: K1801;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA723601) at 1/5,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Immunocytochemistry analysis of RAW264.7 cells untreated / treated with 100ng/mL LPS for 4 hours, add 1μg/mL BFA for last 3 hours labeling Osteopontin with Rabbit anti-Osteopontin antibody (HA723601) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Osteopontin antibody (HA723601) at 1/2,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.