Anti-Synaptophysin antibody [YE269] - Synaptic marker - Mouse IgG1 (Chimeric)

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Synaptophysin antibody [YE269] - Synaptic marker - Mouse IgG1 (Chimeric) (AB309493)

Immunohistochemical analysis of paraffin-embedded human pancreas tissue labeling Synaptophysin with ab309493 at 1/20000 (0.047 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on the islet in human pancreas. The section was incubated with ab309493 for 30 mins at room temperature, followed by anti-mouse IgG1 antibody (ab125913) for 8 mins during the LeicaDS9800 kit staining procedure. 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Synaptophysin antibody [YE269] - Synaptic marker - Mouse IgG1 (Chimeric) (AB309493)

Composite multiplex immunofluorescence staining of SYP, MAP2 and Tau (MBD region) staining in a section of formalin-fixed paraffin-embedded human Alzheimer’s brain*.

Performed on a Leica BOND. The section was pre-treated using heat mediated antigen retrieval with EDTA (Ph9.0) using retrieval settings of 100°C for 40 minutes. The section was then incubated at room temperature for 1 hour with ab309493 at 1µg/ml dilution (shown in green), ab318993 at 1µg/ml (shown in magenta), and ab308439 at 1µg/ml (shown in yellow). Then incubated for 1 hour with ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed 1/1000, ab150086 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 555) preadsorbed 1/1000, and ab150173 Goat Anti-Chicken IgY H&L (Alexa Fluor® 488) preadsorbed 1/1000. Nuclear DNA was labelled with DAPI (shown in blue). The section was then mounted using Dako Fluorescence Mounting Medium ^®.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.

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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Synaptophysin antibody [YE269] - Synaptic marker - Mouse IgG1 (Chimeric) (AB309493)

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling Synaptophysin with ab309493 at 1/20000 (0.047 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Low expression tissue : almost no staining on human kidney. The section was incubated with ab309493 for 30 mins at room temperature, followed by anti-mouse IgG1 antibody (ab125913) for 8 mins during the LeicaDS9800 kit staining procedure. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Synaptophysin antibody [YE269] - Synaptic marker - Mouse IgG1 (Chimeric) (AB309493)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse primary neurons labelling Synaptophysin with ab309493 at 1/100 dilution, followed by ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in mouse primary neuron cell line. Confocal scanning Z step was set as 0.3 µM followed by image processing with maximum Z projection. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). ab183830 Anti-MAP2 rabbit monoclonal antibody was used to counterstain tubulin at 1/1000 dilution, followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) at a 1/500 dilution (Red). The nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Synaptophysin antibody [YE269] - Synaptic marker - Mouse IgG1 (Chimeric) (AB309493)

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling Synaptophysin with ab309493 at 1/20000 (0.047 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on mouse cerebrum. The section was incubated with ab309493 for 30 mins at room temperature, followed by anti-mouse IgG1 antibody (ab125913) for 8 mins during the LeicaDS9800 kit staining procedure. 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Synaptophysin antibody [YE269] - Synaptic marker - Mouse IgG1 (Chimeric) (AB309493)

Composite multiplex immunofluorescence staining of ab302487, ab309493 and ab254351 staining MAP2, Synaptophysin and SV2A in Mouse Primary Neurons DIV14 cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal donkey serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab289874 (shown in yellow), ab309493 (shown in green) and ab254351 (shown in magenta) at 5µg/ml. Cells were then incubated with ab150136 Donkey Anti-Goat IgG H&L (Alexa Fluor® 594) preadsorbed, ab150111 Donkey Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed and ab150061 Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution. Nuclear DNA was labelled with DAPI (shown in blue).

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

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Synaptophysin antibody [YE269] - Synaptic marker - Mouse IgG1 (Chimeric) (AB309493)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized rat primary neurons labelling Synaptophysin with ab309493 at 1/100 dilution, followed by ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in rat primary neuron cell line. Confocal scanning Z step was set as 0.3 µM followed by image processing with maximum Z projection. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). ab183830 Anti-MAP2 rabbit monoclonal antibody was used to counterstain tubulin at 1/1000 dilution, followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) at a 1/500 dilution (Red). The nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Synaptophysin antibody [YE269] - Synaptic marker - Mouse IgG1 (Chimeric) (AB309493)

Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue labeling Synaptophysin with ab309493 at 1/20000 (0.047 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on rat cerebrum. The section was incubated with ab309493 for 30 mins at room temperature, followed by anti-mouse IgG1 antibody (ab125913) for 8 mins during the LeicaDS9800 kit staining procedure. 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• WB

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Western blot - Anti-Synaptophysin antibody [YE269] - Synaptic marker - Mouse IgG1 (Chimeric) (AB309493)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. Performed under reducing conditions. In Western blot, ab309493 was shown to bind specifically to Synaptophysin (SYP).  Target of interest was observed at 38 kDa wild-type 293T cell lysates (lane 1) with no signal observed at this size in SYP knockout cell line (lane 2, knockout cell line ab267272 / knockout cell lysate ab257060). In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 59 seconds.

All lanes:

Western blot - Anti-Synaptophysin antibody [YE269] - Synaptic marker - Mouse IgG1 (Chimeric) (ab309493) at 1/1000 dilution

Lane 1:

Wild-type 293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

Western blot - Human SYP (Synaptophysin) knockout HEK-293T cell lysate (<a href='/en-us/products/cell-lysates/human-syp-synaptophysin-knockout-hek-293t-cell-lysate-ab257060'>ab257060</a>) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG Fc (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-fc-hrp-preadsorbed-ab98717'>ab98717</a>) at 1/20000 dilution

Observed band size: 38 kDa

false

• WB

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Western blot - Anti-Synaptophysin antibody [YE269] - Synaptic marker - Mouse IgG1 (Chimeric) (AB309493)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. Low expression tissue : Kidney (PMID : 11604140). In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 8 seconds.

All lanes:

Western blot - Anti-Synaptophysin antibody [YE269] - Synaptic marker - Mouse IgG1 (Chimeric) (ab309493) at 1/1000 dilution

Lane 1:

Human cerebellum tissue lysate at 10 µg

Lane 2:

Human kidney tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG Fc (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-fc-hrp-preadsorbed-ab98717'>ab98717</a>) at 1/20000 dilution

Observed band size: 38 kDa

false

• WB

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Western blot - Anti-Synaptophysin antibody [YE269] - Synaptic marker - Mouse IgG1 (Chimeric) (AB309493)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. Low expression tissues : Kidney and heart (PMID : 11604140). In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution. Exposure time : 1 second.

All lanes:

Western blot - Anti-Synaptophysin antibody [YE269] - Synaptic marker - Mouse IgG1 (Chimeric) (ab309493) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse cerebellum tissue lysate at 20 µg

Lane 3:

Mouse hippocampus tissue lysate at 20 µg

Lane 4:

Mouse heart tissue lysate at 20 µg

Lane 5:

Mouse kidney tissue lysate at 20 µg

Lane 6:

Rat brain tissue lysate at 20 µg

Lane 7:

Rat cerebellum tissue lysate at 20 µg

Lane 8:

Rat heart tissue lysate at 20 µg

Lane 9:

Rat kidney tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG Fc (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-fc-hrp-preadsorbed-ab98717'>ab98717</a>) at 1/20000 dilution

Observed band size: 38 kDa

false