Immunocytochemistry/ Immunofluorescence analysis of mouse primary neuron cells labeling Synaptophysin with purified ab32127 at 1/100 (2.7µg/mL). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.
Confocal scanning Z step was set as 0.3 µm followed by image processing with maximum Z projection.

All lanes : Anti-Synaptophysin antibody [YE269] (ab32127) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : SYP knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 34 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab32127 observed at 38 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab32127 was shown to react with Syp in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab255356 (knockout cell lysate ab263862) was used. Wild-type HEK-293T and SYP knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab32127 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical staining of paraffin embedded mouse cerebral cortex with purified ab32127 at a dilution of 1/400.

A pre-diluted HRP polymer for rabbit/mouse IgG was used as the secondary antibody and the sample was counterstained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

ab32127 staining Synaptophysin in primary hippocampal rat neurons/glia, (obtained from Neuromics, cat. no. PC35101), DIV14. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab32127 at 0.1µg/ml and ab192757, Mouse mono Anti-PSD95 antibody [K28/43] - Synaptic Marker. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

All lanes : Anti-Synaptophysin antibody [YE269] (ab32127) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate at 20 µg
Lane 2 : SYP knockout HEK-293T cell lysate at 20 µg
Lane 3 : Human brain tissue lysate

Performed under reducing conditions.

Predicted band size: 34 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab32127 observed at 38 kDa. Red - loading control, ab7291 observed at 50 kDa.

ab32127 Anti-Synaptophysin antibody [YE269] was shown to specifically react with Synaptophysin in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab267272 (knockout cell lysate ab257060) was used. Wild-type and Synaptophysin knockout samples were subjected to SDS-PAGE. ab32127 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti- Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti- Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

Immunofluorescent staining of PC-12 (rat adrenal gland pheochromocytoma cell line) cells (fixed in 4% PFA, permeabilized with 0.1% Triton X 100) using purified ab32127 at a dilution of 1/50.

An Alexa Fluor^® 488 goat anti-rabbit antibody was used as the secondary at a dilution of 1/500 and the cells were counterstained with DAPI.

The negative control is shown in the bottom right hand panel - for the negative control, Alex Fluor^® 594 goat anti-mouse was used at a dilution of 1/500.

All lanes : Anti-Synaptophysin antibody [YE269] (ab32127) at 1/100000 dilution (purified)

Lane 1 : Mouse brain
Lane 2 : Rat brain

Secondary
All lanes : HRP goat anti-rabbit at 1/1000 dilution

Predicted band size: 34 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST

Unpurified ab32127 showing positive staining in medulloblastoma tissue.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemical staining of paraffin embedded rat cerebral cortex with purified ab32127 at a dilution of 1/400.

A pre-diluted HRP polymer for rabbit/mouse IgG was used as the secondary antibody and the sample was counterstained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunohistochemical staining of paraffin embedded human pancreas with purified ab32127 at a dilution of 1/400.

A pre-diluted HRP polymer for rabbit/mouse IgG was used as the secondary antibody and the sample was counterstained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Anti-Synaptophysin antibody [YE269] (ab32127) at 1/100000 dilution (purified) + Human fetal brain at 20 µg

Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 34 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?

Anti-Synaptophysin antibody [YE269] (ab32127) at 1/10000 dilution (unpurified) + PC-12 (rat adrenal gland pheochromocytoma cell line) cell lysate

Predicted band size: 34 kDa
Observed band size: 34 kDa
Additional bands at: 21 kDa, 22 kDa. We are unsure as to the identity of these extra bands.

Unpurified ab32127 showing positive staining in lung neuroendocrine tumor tissue.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.