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.

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

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.

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

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.

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-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

All lanes : Anti-PSD95 antibody [EP2652Y] (ab76115) at 1/2000 dilution

Lane 1 : Human brain tissue lysate

Lane 2 : Mouse brain tissue lysate

Lane 3 : Rat brain tissue lysate

Lysates/proteins at 10 µg per lane.

Secondary

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

Predicted band size: 95 kDa

Observed band size: 95 kDa

All lanes : Anti-Synapsin I antibody - Synaptic Marker (ab64581) at 1 µg/ml

Lane 1 : Spinal Cord (Mouse) Tissue Lysate

Lane 2 : Brain (Mouse) Tissue Lysate

Lane 3 : Brain (Rat) Tissue Lysate - normal tissue

Lysates/proteins at 10 µg per lane.

Secondary

All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size: 74 kDa

Observed band size: 70,74 kDa

The immunogen for ab64581 is predicted to recognise both the 70 kDa and 74 kDa isoforms of Synapsin I. Synapsin I is also known to contain a number of potential phosphorylation and glycosylation sites (SwissProt data).