Anti-PSD95 antibody [6G6-1C9] - Synaptic Marker (ab2723)

IHC image of ab2723 staining in formalin-fixed, paraffin-embedded rat retinal tissue, performed on a Leica BOND^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab2723, 2µg/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 hematoxylin and mounted with DPX.

Secondary-only control image is shown as insert.

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.

Lanes 1-6 : Anti-PSD95 antibody [6G6-1C9] - Synaptic Marker (ab2723) at 1/1200 dilution
Lanes 7-12 : Anti-PSD95 antibody [6G6-1C9] - Synaptic Marker (ab2723) at 1/1200 dilution (18 hours at 4°C)

Lanes 1-6 : Mouse Hippocampus with Li-Cor Block Buffer, 1 hour at 21°C
Lanes 7-12 : Mouse Liver with Li-Cor Block Buffer, 1 hour at 21°C

Lysates/proteins at 30 µg per lane.

Secondary
All lanes : Li-Cor IRDye® Donkey anti-mouse 680LT at 1/18000 dilution

Performed under reducing conditions.

Predicted band size: 80 kDa
Additional bands at: 95 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 5 minutes

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ab2723 staining cultured rat primary hippocampal neurons by ICC/IF.

The cultured neurons were fixed with 4% formaldehyde for 5 minutes and blocked with 10% donkey serum in 0.1% PBS- 0.3% TritonX for 30 minutes at 24°C. Cultured neurons were then stained with ab2723 at 1/1000 in 0.3% TritonX with 0.1x PBS and 10% donkey serum for 4h at 24°C. An Alexa-Fluor^® 568 donkey anti-mouse polyclonal antibody at 1/1000 was used as the secondary antibody. PSD95 (which stains the axons and dendrytic spines) can be observed in red (Alexa-Fluor^® 568 secondary). Synaptophysin is observable in green (Alexa-Fluor^® 488 secondary) and co-localizes with PSD95 in dendritic spines. Nuclei are stained in blue with 1.43µM Hoechst.

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All lanes : Anti-PSD95 antibody [6G6-1C9] - Synaptic Marker (ab2723) at 5 µg/ml

Lane 1 : Hippocampus (Rat) Tissue Lysate
Lane 2 : Forebrain (Rat) Tissue Lysate
Lane 3 : Hippocampus (Mouse) Tissue Lysate
Lane 4 : Forebrain (Mouse) Tissue Lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 80 kDa
Observed band size: 95 kDa why is the actual band size different from the predicted?
Additional bands at: 100 kDa, 110 kDa, 80 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 3 minutes

ab2723 staining PSD95 in zebrafish retina (outer plexiform layer) tissue sections by Immunohistochemistry (IHC-Fr - frozen sections).

Tissue was fixed with paraformaldehyde, permeabilized with Triton X-100 and blocked with 5% BSA for 1 hour at 23°C. Sodium citrate antigen retrieval was used. Samples were incubated with primary antibody (1/100) for 16 hours at 4°C. An Alexa-Fluor^® 647-conjugated goat anti-mouse IgG polyclonal (1/1000) was used as the secondary antibody.

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ab2723 at 1/200 staining rat primary hippocampal neurons by ICC/IF.

The cells were paraformaldehyde fixed and then permeabilized with 0.1% Triton X-100 before being stained with the antibody for 1 hour. An Alexa-Fluor ^® 594 conjugated goat polyclonal antibody was used as the secondary.

The antibody shows numerous puncta along the shafts.

 

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IHC-FoFr staining of PSD95 on rat hippocampus section using ab2723 (1/3000).

The sections used came from animals perfused fixed with 4% paraformaldehyde with 15% of a solution of saturated picric acid, in phosphate buffer 0.1M. Following post-fixation in the same fixative overnight, the brains were cryoprotected in sucrose 30% overnight. Brains were then cut using a cryostat and the immunostainings were performed using the ‘free floating’ technique.

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