重组Anti-PGP9.5抗体[EPR4118] - Neuronal Marker (ab108986)

Flow cytometry overlay histogram showing left Neuro2a positive cells and right negative NIH3T3 stained with ab108986 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab108986) (1x 10⁶ in 100μl at 0.2μg/ml (1/10500)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in Neuro2a Fixed with 80% methanol (5 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

Lanes 1 - 5: Merged signal (red and green). Green - ab108986 observed at 25 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab108986 was shown to react with PGP9.5 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab255443 (knockout cell lysate ab263773) was used. Wild-type and PGP9.5 knockout samples were subjected to SDS-PAGE. ab108986 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4^°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Lanes 1 - 4: Merged signal (red and green). Green - ab108986 observed at 24 kDa. Red - loading control, ab8245, observed at 37 kDa.

Ab108986 was shown to specifically react with UCHL1 in wild-type cells as signal was lost in UCHL1 knockout HAP1 cells. Wild-type and UCHL1 knockout samples were subjected to SDS-PAGE.  Ab108986 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 dilution respectively. Blots were developed 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 1/10000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Frozen sections) analysis of mouse cerebrum tissue sections labeling PGP9.5 with Purified ab108986 at 1/250 (0.5 µg/ml).Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. DAPI was used as a counterstain.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Neuro-2a (Mouse neuroblastoma cell line) cells labeling PGP9.5 with ab108986 at 1/500 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on Neuro-2a cell line. The nuclear counter stain is DAPI (blue).

The negative control is PBS only.

Immunohistochemical analysis of paraffin-embedded human hepatocellular carcinoma tissue labeling PGP9.5 with ab108986, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining on human hepatocellular carcinoma. The section was incubated with ab229902 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Y79 (Human retinoblastoma retinoblastoma)cells labelling PGP9.5 with ab108986 at 1/20 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) isotype control (black)and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluorr^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. 

PGP9.5 was immunoprecipitated from 0.35 mg Human fetal brain lysate  with ab108986 at 1/20 dilution (0.5μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab108986 1/500 dilution (0.17 μg/ml). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used as the secondary antibody at 1/1000 dilution.

Lane 1: Human fetal brain lysate 10μg
Lane 2: ab108986 IP in Human fetal brain lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab108986 in Human fetal brain lysate.

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

Exposure time: 1 second.

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

Immunohistochemical analysis of paraffin-embedded rat cerebral cortex tissue labeling PGP9.5 with ab108986, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining on rat cerebral cortex.The section was incubated with ab229902 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Immunohistochemical analysis of paraffin-embedded mouse cerebral cortex tissue labeling PGP9.5 with ab108986, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining on mouse cerebral cortex. The section was incubated with ab229902 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Formalin-fixed, paraffin-embedded Cat lung tissue stained for PGP9.5 using ab108986 at 1/250 dilution in immunohistochemical analysis. The secondary antibody was ImmPRESS™ HRP Universal Antibody (Anti-Mouse IgG/A). Antigen retrieval: Heat mediated - Buffer/Enzyme Used: 10 mM citrate, pH6.0.

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Overlay histogram showing SH-SY5Y cells stained with ab108986 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab108986, 1/10000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluorr^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in SH-SY5Y cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions. 

Immunohistochemical analysis of rat Jejunum tissue sections labeling PGP9.5 with ab108986 at a dulution of 1/1000. Sections were fixed with Formaldehyde. A Biotin conjugated Goat Anti-Rabbit IgG at 1/300 was used as the secondary antibody. Antigen retrieval was heat mediated using citric acid. 

All nerve components of enteric plexuses appear to be very well demonstrated, particularly the fine fibres of the lamina propria and the muscularis mucosa.

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Fomalin-fixed, paraffin-embedded Dog skin tissue stained for PGP9.5 using ab108986 at 1/500 dilution in immunohistochemical analysis. ImmPRESS™ Anti-Rabbit IgG Polymer Detection Kit was used as the secondary antibody.

Antigen Retrieval: Heat mediated - Buffer/Enzyme Used: 10 mM citrate, pH6.0

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ab108986 staining PGP9.5 in human colon tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 2% BSA for 10 minutes at 21°C; antigen retrieval was by heat mediation in citric acid. Samples were incubated with the primary antibody (1/500 in TBS/BSA/azide) for 16 hours at 21°C. A Biotin-conjugated goat anti-rabbit IgG polyclonal (1/300) was used as the secondary antibody.

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Immunohistochemical analysis of mouse colon tissue sections labeling PGP9.5 with ab108986 at a dulution of 1/1500. Sections were fixed with Formaldehyde. A Biotin conjugated Goat Anti-Rabbit IgG at 1/300 was used as the secondary antibody. Antigen retrieval was heat mediated using citric acid. 

All nerve cell/fibre components of enteric plexuses are demonstrated very well.

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