Anti-TPX2 antibody [18D5-1] (ab32795)

  Immunocytochemistry/ Immunofluorescence - TPX2 antibody [18D5-1] (ab32795)

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.

This image was kindly submitted by Serena Orlando, Giulia Guarguaglini and Patrizia Lavia, University 'La Sapienza' CNR, Italy.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - TPX2 antibody [18D5-1] (ab32795)

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.