ab15440 detecting Nucleophosmin in interphase mouse primary embryonic fibroblasts. Cells were also counterstained with anti gamma tubulin antibody and DAPI in order to highlight the centrossomes and the nucleus (respectively). Secondary antibodies: goat anti-rabbit IgG conjugated to Alexa 488 (for ab15440) and goat anti-mouse IgG conjugated to Alexa 594 (for gamma-tubulin antibody).

ab15440 detecting Nucleophosmin in mitotic mouse primary embryonic fibroblasts. Cells were also counterstained with anti gamma tubulin antibody and DAPI in order to highlight the centrossomes and the nucleus (respectively). Secondary antibodies: goat anti-rabbit IgG conjugated to Alexa 488 (for ab15440) and goat anti-mouse IgG conjugated to Alexa 594 (for gamma-tubulin antibody).

ab15440 (4µg/ml) staining nucleophosmin in human colon using an automated system (DAKO Autostainer Plus). Using this protocol there is nuclear staining.
Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffer citrate pH 6.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human kidney tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1/500 with a rabbit polyclonal antibody recognizing Nucleophosmin (ab15440) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.