All lanes : Anti-PSD95 antibody - Synaptic Marker (ab18258) at 1 µg/ml

Lane 1 : Brain (Mouse) Tissue Lysate (ab27253)
Lane 2 : Brain (Rat) Tissue Lysate (ab7942)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Rabbit IgG secondary antibody (ab28446) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 80 kDa
Observed band size: 85 kDa why is the actual band size different from the predicted?

ab18258 (1/500) staining PSD95 in paraffin-embedded mouse cerebellum (top) and medulla (bottom) tissue, showing positive staining to the synaptic regions of the brain. Tissue was fixed in formaldehyde, blocking performed using 1% BSA (10 minutes/RT) and heat mediated antigen retrieval performed before staining. The secondary antibody (1/200) was goat anti rabbit IgG conjugated to Biotin.  For further experimental details, please refer to abreview.

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ab18258 staining PSD95 in SH-SY5Y cells. The cells were fixed with 100% methanol (5 min) at room temperature, permeabilized with 0.1% Triton X-100 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 with the antibody ab18258 at 5µg/ml and ab7291 (Mouse monoclonal to alpha Tubulin - Loading Control) used at a 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed, (pseudo-colored green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594) preadsorbed, (colored red), both used at a 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature.

ab18258 (1/1000) staining PSD9 in paraffin-embedded rat cerebellum. Tissue was fixed in formaldehyde, blocking performed using 1% BSA (10 minutes/RT) and heat mediated antigen retrieval performed before staining. The secondary antibody (1/200) was goat anti rabbit IgG conjugated to Biotin. For further experimental details, please refer to abreview.

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IHC image of PSD95 staining in mouse brain 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 with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 minutes. The section was then incubated with ab18258, 1 µg/ml, for 15 minutes 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.

ab18258 staining PSD95 in human SH-SY5Y cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed in paraformaldehyde, blocked with 10% serum for 20 minutes at 24°C, then incubated with ab18258 at a 1/1000 dilution for 16 hours at 4°C. The secondary used was an Alexa-Fluor^® 488 conjugated donkey anti-rabbit polyclonal used at a 1/1000 dilution. Counterstained with Hoechst 33258 (blue).

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