Recombinant Anti-PGP9.5 antibody [EPR4118] - BSA and Azide free (ab220823)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

Clone EPR4118 (ab220823) has been successfully conjugated by Abcam. This image was generated using Anti-PGP9.5 antibody [EPR4118] (Alexa Fluor® 647). Please refer to ab196173 for protocol details.

IHC image of PGP9.5 staining in a section of formalin-fixed paraffin-embedded normal human pancreas*.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) in a Dako Pascal pressure cooker using the standard factory-set regime. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 1% (w/v) BSA for 1h at room temperature. The section was then incubated overnight at +4°C in TBS containing 0.025% (v/v) Triton X-100 and 1% (w/v) BSA with ab196173 at 1/100 (shown in red) and counterstained using ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue). The section was then mounted using Fluoromount^®.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

This ICC/IF data was generated using the same anti-PGP9.5 antibody clone, EPR4118, in a different buffer formulation (cat# ab108986).

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.

This data was developed using the same antibody clone in a different buffer formulation (ab108986).

Lanes 1-4: 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 in western blot. Loss of signal was observed when knockout sample 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.

This WB data was generated using the same anti-PGP9.5 antibody clone, EPR4118, in a different buffer formulation (cat# ab108986).

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 (KO) in wild-type cells as signal was lost in UCHL1 (KO) knockout HAP1 cells. Wild-type and UCHL1 (KO) 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.

This data was generated using the same anti-PGP9.5 antibody clone, EPR4118, in a different buffer formulation (cat# ab108986).

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).

This data was generated using the same anti-PGP9.5 antibody clone, EPR4118, in a different buffer formulation (cat# ab108986).

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 Fluor^®488, ab150077) at 1/2000 dilution was used as the secondary antibody. 

This data was generated using the same anti-PGP9.5 antibody clone, EPR4118, in a different buffer formulation (cat 

ab108986). 

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.

This data was generated using the same anti-PGP9.5 antibody clone, EPR4118, in a different buffer formulation (cat# ab108986).

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).

This data was generated using the same anti-PGP9.5 antibody clone, EPR4118, in a different buffer formulation (cat# ab108986).

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).

This data was generated using the same anti-PGP9.5 antibody clone, EPR4118, in a different buffer formulation (cat# ab108986).

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 Fluor^® 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. 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab108986). 

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab108986).

See Abreview

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab108986).

See Abreview

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab108986).

See Abreview

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab108986).