Anti-Synaptophysin antibody [YE269] - Synaptic Marker

• WB

Lab

Western blot - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (AB32127)

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.

All lanes:

Western blot - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (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 2:

Western blot - Human SYP (Synaptophysin) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-syp-synaptophysin-knockout-hek-293t-cell-line-ab267272'>ab267272</a>)

Lane 3:

Human brain tissue lysate

Predicted band size: 33 kDa

Observed band size: 38 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (AB32127)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (AB32127)

Immunohistochemical analysis of formalin fixed paraffin embedded human pancreas labelling synaptophysin with ab32127 at a concentration of 1 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab32127 Anti-Synaptophysin antibody [YE269] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (AB32127)

Immunohistochemical analysis of paraffin-embedded human Alzheimer's brain tissue* labelling SYP with ab32127 at 1ug/ml followed by a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

Positive staining in human Alzheimer's brain.

The section was incubated with ab32127 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

Heat mediated antigen retrieval with sodium citrate buffer (pH 6.0 epitope retrieval solution1) for 20 mins.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (AB32127)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (AB32127)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (AB32127)

ab32127 staining Synaptophysin in primary mouse neurons/glia, DIV14 (prepared from E18 mouse hippocampal brain area, obtained from Transnetyx Tissue by BrainBits, LLC, cat.no. C57EHP) cells. The cells were fixed with 4% paraformaldehyde (10 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 100% methanol (5 min).

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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (AB32127)

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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (AB32127)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (AB32127)

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.

• WB

Lab

Western blot - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (AB32127)

All lanes:

Western blot - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (ab32127) at 1/100000 dilution

All lanes:

Human fetal brain at 20 µg

Secondary

All lanes:

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

Predicted band size: 33 kDa

Observed band size: 38 kDa

false

• WB

Unknown

Western blot - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (AB32127)

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.

All lanes:

Western blot - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (ab32127) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

Syp knockout HEK293T cell lysate at 20 µg

Lane 3:

Human brain tissue lysate at 20 µg

Predicted band size: 33 kDa

Observed band size: 38 kDa

false

• WB

Lab

Western blot - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (AB32127)

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.

All lanes:

Western blot - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (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 2:

Western blot - Human SYP (Synaptophysin) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-syp-synaptophysin-knockout-hek-293t-cell-line-ab255356'>ab255356</a>)

Predicted band size: 33 kDa

Observed band size: 38 kDa

false

• WB

Unknown

Western blot - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (AB32127)

Blocking/Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (ab32127) at 1/100000 dilution

Lane 1:

Mouse brain

Lane 2:

Rat brain

Secondary

All lanes:

HRP goat anti-rabbit at 1/1000 dilution

Predicted band size: 33 kDa

Observed band size: 38 kDa

false

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (AB32127)

Unpurified ab32127 showing positive staining in medulloblastoma tissue.

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (AB32127)

Unpurified ab32127 showing positive staining in lung neuroendocrine tumor tissue.

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

• WB

Unknown

Western blot - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (AB32127)

All lanes:

Western blot - Anti-Synaptophysin antibody [YE269] - Synaptic Marker (ab32127) at 1/10000 dilution

All lanes:

PC-12 (rat adrenal gland pheochromocytoma cell line) cell lysate

Predicted band size: 33 kDa

Observed band size: 21 kDa,22 kDa,34 kDa

false