Synaptic Marker (Synaptophysin, Synapsin1, PSD95, VAMP2) Antibody Sampler Panel - Human, Mouse (ab263463)

ab181869 staining VAMP2 in U87-MG (human glioblastoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 100% methanol. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. DAPI was used as a nuclear counterstain.

Negative control 1:PBS only.

Immunohistochemical analysis of paraffin-embedded Human hippocampus tissue labeling Synapsin-1 with ab254349 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining in human hippocampus (PMID: 22900032). The section was incubated with ab254349 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Lane1:
Primary: ab254349, 1/1000 dilution
Sample: Human brain tissue lysate, 20 ug
Secondary: VeriBlot for IP secondary antibody(HRP)(ab131366), 1/1000 dilution

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

This blot was developed using a higher sensitivity ECL substrate.

Exposure time: 15 seconds.

Observed band: 75/70 kDa

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse retina tissue labeling Synapsin-1 with ab254349 at 1/100 dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 (2 ug/ml) dilution (Green). Positive staining on mouse retina is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 (2 ug/ml) dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 0.1% Tween-20 permeabilized Mouse primary brain cells cells labelling Synapsin-1 with ab254349 at 1/50 dilution (1ug) (Right) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Left). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. 

Synapsin-1 was immunoprecipitated from 0.35 mg Mouse brain tissue lysate with ab254349 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254349 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/1000 dilution.

Lane 1: Mouse brain tissue lysate 10 ug

Lane 2: ab254349 IP in Mouse brain tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254349 in mouse brain tissue lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 1 second.

Observed band: 70/75 kDa

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized mouse primary neuron cells labelling Synapsin-1 with ab254349 at 1/500 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing positive staining in mouse primary neuron. Confocal scanning Z step was set as 0.3 μm followed by image processing with maximum Z projection. ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain MAP2 at 1/500 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) at 1/1000 dilution (Red). The Nuclear counterstain was DAPI (Blue).

-ve control 1: ab254349 at 1/500 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) at 1/1000 dilution.

-ve control 2: ab11267 at 1/500 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution.

Lanes 1 - 4: Merged signal (red and green). Green - ab238135 at 1/2000 dilution observed at 75 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa. Secondary antibodies: Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776), 1/20000 dilution

Lane 1: Wild-type SH-SY5Y cell lysate, 20 ug

Lane 2: DLG4 knockout SH-SY5Y cell lysate, 20 ug

Lane 3: HeLa cell lysate, 20 ug

Lane 4: HL-60 cell lysate, 20 ug

ab238135 was shown to react with PSD95 in wild-type SH-SY5Y cells in Western blot with loss of signal observed in DLG4 knockout cell line ab280043 (DLG4 knockout cell lysate ab280102). Wild-type SH-SY5Y and DLG4 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab238135 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h 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.

ab192757 stained U-87 MG cells. The cells were 4% formaldehyde fixed for 10 minutes at room temperature and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab192757 at 5µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (ab150117) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

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.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse cerebrum tissue labeling Synapsin-1 with ab254349 at 1/100 dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 (2 ug/ml) dilution (Green). Positive staining on mouse cerebrum is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 (2 ug/ml) dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

IHC image of PSD95 [K28/43] staining in Human normal cerebral cortex formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval (EDTA based pH 9.0 solution, epitope retrieval solution 2) for 20 mins. The section was then incubated with ab192757, 0.05µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

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

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

Western blotting with ab238135 at 1/2000 dilution. ab97051 was used as a secondary at 1/100000 dilution.

Lane 1: Mouse brain lysate 20 µg

Lane 2: Mouse cerebellum lysate 20 µg

Lane 3: Rat brain lysate 20 µg

Blocking and diluting buffer and concentration: 5% NFDM/TBST

The molecular weight observed is consistent with what has been described in the literature (PMID: 20118925).

Exposure time: 15 seconds

Observed band: 75 and 100 kDa

Immunohistochemical analysis of paraffin-embedded Mouse pancreas tissue labeling Synapsin-1 with ab254349 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining in mouse pancreatic islets (PMID: 22334712). The section was incubated with ab254349 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Western blotting with ab238135 at 1/2000 dilution. ab131366 was used as a secondary at 1/1000 dilution.

Lane 1: Human cerebellum lysate, 20 ug

Lane 2: Human brain lysate, 20 ug

Blocking and diluting buffer and concentration: 5% NFDM/TBST

The molecular weight observed is consistent with what has been described in the literature (PMID: 20118925)

Exposure time: 48 seconds

Observed band: 75 and 100 kDa

Anti-VAMP2 antibody [EPR12790] (ab181869) at 1/50000 dilution (purified) + Mouse brain lysate at 10 µg

Secondary

Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 13 kDa

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

All lanes : Anti-PSD95 antibody [K28/43] (ab192757) at 1 µg/ml

Lane 1 : Human brain tissue lysate - total protein (ab29466)

Lane 2 : Brain (Mouse) Tissue Lysate

Lane 3 : Brain (Rat) Tissue Lysate

Lane 4 : Human Cerebral Cortex Tissue Lysate

Lysates/proteins at 10 µg per lane.

Secondary

All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 80 kDa

Observed band size: 80,95 kDa

Exposure time: 30 seconds

Human medulloblastoma labelled with ab52636 at 1/250 - 1/500 dilution.