ab32795 staining TPX2 in HeLa cells and mouse NIH-3T3 cells (fuzzier pattern, different from the high-quality sharp signal seen in Human cells), by immunofluorescence.
optimal antibody dilution: 4µg/ml
optimal fixation protocol: PFA/Triton fixation: 10 min room at room temperature, in 3,7 % PFA diluted in PHEM buffer (45  mM Hepes pH 6,9, 45 mM Pipes pH 6,9, 5 mM MgCl2, 10 mM EGTA) containing 0.2% Triton X-100, followed  by 3 washes in PBS    -  Alternative fixation protocol also gives good staining: 6 min in cold Methanol at -20°C,  then 3 washes in PBS.
IF was performed following a standard protocol: Blocking, 30 min; primary antibody, 1 hr; secondary antibody, 45 min. All incubations were at 37 °C in PBS/ 0.1% Tween containing 3% BSA.

ab32795 (1µg/ml) staining TPX2 in human testis using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear and cytoplasmic staining .
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH6.1 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min 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.

Immunofluorescent analysis of PLK1 using PLK1 Monoclonal antibody (13E8) ab32795 shows staining in WiDr colon carcinoma cells. PLK1 staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing PLK1 ab32795 at a dilution of 1:20 over night at 4 ?C washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

Immunofluorescent analysis of PLK1 using PLK1 Monoclonal antibody (13E8) ab32795 shows staining in HeLa cells. PLK1 staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing PLK1 ab32795 at a dilution of 1:20 over night at 4 ?C washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

Immunofluorescent analysis of PLK1 using PLK1 Monoclonal antibody (13E8) ab32795 shows staining in U251 glioma cells. PLK1 staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing PLK1 ab32795 at a dilution of 1:20 over night at 4 ?C washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

Immunohistochemistry was performed on biopsies of deparaffinized Human testis 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 1:200 with a mouse monoclonal antibody recognizing TPX2 ab32795 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.