Immunocytochemistry/Immunofluorescence staining of Neuro-2a (mouse neuroblastoma) cells labelling TPPP with purified ab92305 at a working dilution of 1/100. The secondary antibody was Alexa Fluor® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. DAPI was used as nuclear counterstain. The cells were fixed in 4% Paraformaldehyde and permeabilized using 0.1% Triton X-100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, rabbit primary antibody was used followed by an Alexa Fluor® 594 goat anti-mouse antibody (ab150120). For negative control 2, ab7291 (mouse anti-tubulin) was used followed by an Alexa Fluor® 488 goat anti-rabbit secondary (ab150077).

All lanes : Anti-TPPP antibody [EPR3316] (ab92305) at 1/10000 dilution

Lane 1 : Human cerebellum tissue lysate
Lane 2 : Mouse brain tissue lysate
Lane 3 : Mouse cerebral cortex tissue lysate
Lane 4 : Rat brain tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

Predicted band size: 24 kDa
Observed band size: 25 kDa why is the actual band size different from the predicted?



Blocking/Diluting buffer 5% NFDM /TBST

Immunohistochemical analysis of paraffin-embedded human cerebral cortex tissue sections labelling TPPP with purified ab92305 at dilution of 1/50. The secondary antibody used was ab97051; a goat anti-rabbit IgG H&L (HRP) at dilution of 1/500. The sample was counterstained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

Overlay histogram showing 4% paraformaldehyde fixed Neuro-2a (mouse neuroblastoma) cells labelling TPPP with purified ab92305 at dilution of 1/20. The secondary antibody used was Alexa Fluor® 488 goat-anti-rabbit IgG at dilution of 1/2000. A non-specific IgG antibody (rabbit monoclonal) was used as isotype control (black line). The blue line shows cells without incubation with primary antibody and secondary antibody. 

All lanes : Anti-TPPP antibody [EPR3316] (ab92305) at 1/1000 dilution

Lane 1 : Fetal brain lysate
Lane 2 : SHSY5Y cell lysate
Lane 3 : Mouse brain lysate
Lane 4 : Rat brain lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit antibody at 1/2000 dilution

Predicted band size: 24 kDa

Immunohistochemical analysis of paraffin-embedded human glioma tissue sections labelling TPPP with purified ab92305 at dilution of 1/50. The secondary antibody used was ab97051; a goat anti-rabbit IgG H&L (HRP) at dilution of 1/500. The sample was counterstained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

ab92305 at 1/100 dilution staining TPPP in SH-SY5Y cells, by immunofluorescence.

Immunohistochemical analysis of paraffin-embedded mouse cerebral cortex tissue sections labelling TPPP with purified ab92305 at dilution of 1/50. The secondary antibody used was ab97051; a goat anti-rabbit IgG H&L (HRP) at dilution of 1/500. The sample was counterstained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

Immunohistochemical analysis of paraffin-embedded rat cerebral cortex tissue sections labelling TPPP with purified ab92305 at dilution of 1/50. The secondary antibody used was ab97051; a goat anti-rabbit IgG H&L (HRP) at dilution of 1/500. The sample was counterstained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

Overlay histogram showing SH-SY5Y cells stained with ab92305 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab92305, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

ab92305 at 1/250 dilution staining TPPP in paraffin-embedded Human brain tissue by immunohistochemistry.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.