Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Heme Oxygenase 1 antibody (ab13243)

All lanes : Anti-Heme Oxygenase 1 antibody (ab13243) at 1/1000 dilution

Lane 1 : Hek293
Lane 2 : HL60
Lane 3 : HeLa
Lane 4 : A549
Lane 5 : Hu spleen
Lane 6 : Ms spleen
Lane 7 : Rt spleen

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : IRDye® 800CW Goat anti Rabbit

Predicted band size: 34.6 kDa
Observed band size: 32 kDa why is the actual band size different from the predicted?

Hek293 & HL60 presumed negative or very low expression.

Loading control GAPDH at 38kDa

IHC image of Heme Oxygenase 1 staining in formalin fixed, paraffin embedded rat spleen normal tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab13243, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

 

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Anti-Heme Oxygenase 1 antibody (ab13243) (Rat microsomes)
Predicted band size: 34.6 kDa

Alzheimer diseased section stained with ab13243.

ICC/IF image of ab13243 stained HepG2 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab13243, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Standard Curve for Heme Oxygenase 1 (Analyte: Heme Oxygenase 1 protein (Tagged) (ab85243)); dilution range 1pg/ml to 1µg/ml using Capture Antibody Mouse monoclonal [HO-1-1] to Heme Oxygenase 1 (ab13248) at 5µg/ml and Detector Antibody Rabbit polyclonal to Heme Oxygenase 1 (ab13243) at 0.5µg/ml.