Anti-PSD95 抗体 - Synaptic Marker

• WB

Lab

Western blot - Anti-PSD95 antibody - Synaptic Marker (AB18258)

Western blot : Anti-DLG4 antibody (ab18258) staining at 1 ug/ml, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab18258 was shown to bind specifically to DLG4. A band was observed at 80 kDa in wild-type U-87 MG cell lysates with no signal observed at this size in DLG4 knockout cell line. To generate this image, wild-type and DLG4 knockout U-87 MG cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-PSD95 antibody - Synaptic Marker (ab18258) at 1 µg/mL

Lane 1:

Wild-type U-87 MG cell lysate at 20 µg

Lane 2:

DLG4 knockout U-87 MG cell lysate at 20 µg

Lane 3:

Wild-type SH-SY5Y ab275475 cell lysate at 20 µg

Lane 4:

DLG4 knockout SH-SY5Y <a href='/products/cell-lines/human-psd95-knockout-sh-sy5y-cell-line-ab280043'>ab280043</a> cell lysate at 20 µg

Lane 5:

Human Brain cell lysate at 20 µg

Lane 6:

HL-60 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 80 kDa

false

• IHC-P

AbReview17646****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PSD95 antibody - Synaptic Marker (AB18258)

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.

This image is courtesy of an abreview submitted by Carl Hobbs, King's College London, United Kingdom

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PSD95 antibody - Synaptic Marker (AB18258)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PSD95 antibody - Synaptic Marker (AB18258)

Immunohistochemical analysis of formalin fixed paraffin embedded human cerebellum labelling PSD95 with ab18258 at a concentration of 1 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab18258 Anti-PSD95 antibody was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• IHC-P

Collaborator

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PSD95 antibody - Synaptic Marker (AB18258)

ab18258 (1/1000 dilution) 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 dilution) was goat anti rabbit IgG conjugated to Biotin. For further experimental details please refer to abreview.

This image is courtesy of an abreview submitted by Carl Hobbs, King's College London, United Kingdom.

• ICC/IF

AbReview26168****

Immunocytochemistry/ Immunofluorescence - Anti-PSD95 antibody - Synaptic Marker (AB18258)

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).

Image courtesy of an anonymous Abreview.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PSD95 antibody - Synaptic Marker (AB18258)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PSD95 antibody - Synaptic Marker (AB18258)

IHC image of PSD95 staining in a section of formalin-fixed paraffin-embedded normal rat eye 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 20mins. The section was then incubated with ab18258 1ug/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. The inset secondary-only control image is taken from an identical assay without primary antibody.

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PSD95 antibody - Synaptic Marker (AB18258)

ab18258 staining PSD95 in primary hippocampal rat neurons/glia (obtained from Neuromics cat. no. PC35101) DIV14.cells. The cells were fixed with 100% methanol (5 min) permeabilized with 0.1% PBS-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 overnight at 4°C with ab18258 at 5µg/ml and ab7291 Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081 Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488) pre-adsorbed at 1/1000 dilution (shown in green) and ab150120 Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594) pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

• WB

Lab

Western blot - Anti-PSD95 antibody - Synaptic Marker (AB18258)

Gel type : MOPS

Blocking buffer : 1% milk

All lanes:

Western blot - Anti-PSD95 antibody - Synaptic Marker (ab18258) at 1 µg/mL

Lane 1:

Mouse brain tissue lysate at 10 µg

Lane 2:

Human brain tissue lysate at 20 µg

Lane 3:

SHSY5Y whole cell lysate at 20 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/50000 dilution

Predicted band size: 80 kDa

Observed band size: 85 kDa

false

Exposure time: 8min

• WB

Lab

Western blot - Anti-PSD95 antibody - Synaptic Marker (AB18258)

Western blot : Anti-DLG4 antibody (ab18258) staining at 1 ug/ml, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab18258 was shown to bind specifically to DLG4. A band was observed at 80 kDa in wild-type U-87 MG cell lysates with no signal observed at this size in DLG4 knockout cell line. To generate this image, wild-type and DLG4 knockout U-87 MG cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-PSD95 antibody - Synaptic Marker (ab18258) at 1 µg/mL

Lane 1:

Wild-type U-87 MG cell lysate at 20 µg

Lane 2:

DLG4 knockout U-87 MG cell lysate at 20 µg

Lane 3:

Wild-type SH-SY5Y ab275475 cell lysate at 20 µg

Lane 4:

DLG4 knockout SH-SY5Y <a href='/products/cell-lines/human-psd95-knockout-sh-sy5y-cell-line-ab280043'>ab280043</a> cell lysate at 20 µg

Lane 5:

Human Brain cell lysate at 20 µg

Lane 6:

HL-60 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 80 kDa

false

• WB

AbReview67157****

Western blot - Anti-PSD95 antibody - Synaptic Marker (AB18258)

All lanes:

Western blot - Anti-PSD95 antibody - Synaptic Marker (ab18258) at 1/2000 dilution

All lanes:

Human differentiated neurons whole cell lysate at 5 µg

Secondary

All lanes:

Goat Anti-Rabbit HRP at 1/2000 dilution

true

Exposure time: 1min

This image is courtesy of an anonymous Abreview

• WB

Unknown

Western blot - Anti-PSD95 antibody - Synaptic Marker (AB18258)

All lanes:

Western blot - Anti-PSD95 antibody - Synaptic Marker (ab18258) at 1 µg/mL

Lane 1:

Brain (Mouse) Tissue Lysate (ab27253) at 10 µg

Lane 2:

Brain (Rat) Tissue Lysate (<a href='/products/unavailable/brain-rat-tissue-lysate-ab7942'>ab7942</a>) at 10 µg

Secondary

All lanes:

Rabbit IgG secondary antibody (ab28446) at 1/10000 dilution

Predicted band size: 80 kDa

Observed band size: 85 kDa

false