Anti-PBR antibody [EPR5384]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PBR antibody [EPR5384] (AB109497)

Immunohistochemical analysis of formalin fixed paraffin embedded human adrenal gland labelling PBR with ab109497 at a concentration of 0.05µg/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.

ab109497 anti-PBR antibody [EPR5384] 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

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PBR antibody [EPR5384] (AB109497)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon tissue labelling PBR with unpurified ab109497 at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-PBR antibody [EPR5384] (AB109497)

Overlay histogram showing HepG2 cells stained with unpurified ab109497 (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. The cells were then incubated with the antibody (unpurified ab109497, 1/50 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PBR antibody [EPR5384] (AB109497)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human bladder carcinoma tissue labelling PBR with purified ab109497 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PBR antibody [EPR5384] (AB109497)

ab109497 staining PBR in wild-type HeLa cells (top panel) and TSPO knockout HeLa cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10min), 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 ab109497 at 0.1 μg/ml and ab195884 at 2 μg/ml (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

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

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-PBR antibody [EPR5384] (AB109497)

Intracellular Flow Cytometry analysis of U-87MG cells labelling PBR with purified ab109497 at 1/150 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PBR antibody [EPR5384] (AB109497)

ab109497 staining PBR in HeLa. The cells were fixed with 100% methanol (5min), 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 ab109497 at 1μg/ml and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PBR antibody [EPR5384] (AB109497)

Immunocytochemistry/Immunofluorescence analysis of U-87MG cells labelling PBR with purified ab109497 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

Control 1 : primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2 : ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PBR antibody [EPR5384] (AB109497)

ab109497 staining PBR in wild-type HAP1 cells (top panel) and PBR knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), 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 ab109497 at 1μg/ml and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

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

• IP

Lab

Immunoprecipitation - Anti-PBR antibody [EPR5384] (AB109497)

ab109497 (purified) at 1/60 immunoprecipitating PBR in A431 whole cell lysate.

Lane 1 (input) : A431 whole cell lysate (10μg)

Lane 2 (+) : ab109497 + A431 whole cell lysate (10μg).

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab109497 in A431 whole cell lysate.

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1500 dilution.

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-PBR antibody [EPR5384] (ab109497)

Predicted band size: 19 kDa

Observed band size: 19 kDa

false

• IP

Lab

Immunoprecipitation - Anti-PBR antibody [EPR5384] (AB109497)

ab109497 (purified) at 1/60 immunoprecipitating PBR in U-87MG whole cell lysate.

Lane 1 (input) : U-87MG whole cell lysate (10μg)

Lane 2 (+) : ab109497 + U-87MG whole cell lysate (10μg).

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab109497 in U87-MG whole cell lysate.

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1500 dilution.

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-PBR antibody [EPR5384] (ab109497)

Predicted band size: 19 kDa

Observed band size: 19 kDa

false

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PBR antibody [EPR5384] (AB109497)

TSPO (PBR) expression was abolished in global KO mice without pathological changes

TSPO expression in different tissues from WT and KO mice was detected by western blotting (A) and IHC (B). Scale Bars, 100μm.

4% PFA-fixed tissue sections were blocked with 5% goat serum and incubated overnight at 4°C with ab109497 at 1/1500 dilution. DAB staining.

Note : TSPO and PBR are alternative names for the same target.

(Adapted from Figure 3 of Wang et al)

Wang, H. et al PLoS One. 2016 Dec 1;11(12):e0167307. doi: 10.1371/journal.pone.0167307. eCollection 2016 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• IHC-Fr

Unknown

Immunohistochemistry (Frozen sections) - Anti-PBR antibody [EPR5384] (AB109497)

Immunohistochemistry (Frozen sections) analysis of mouse kidney tissue sections labeling PBR with Purified ab109497 at 1/250 (3.8 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PBR antibody [EPR5384] (AB109497)

Immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections) analysis of mouse hypothalamus tissue labelling PBR with ab109497 at 1/1000 dilution (1.03 μg/ml). Heat mediated antigen retrieval was performed using Tris/EDTA buffer, pH 9.0 (ab93684). A goat anti-rabbit IgG (H+L) (HRP) was used as the secondary antibody (concentration : ready to use). A secondary-antibody-only control was also performed. Counterstained with hematoxylin.

Positive staining was observed on ependymal cells and endothelial cells in mouse hypothalamus.

• IHC-Fr

Unknown

Immunohistochemistry (Frozen sections) - Anti-PBR antibody [EPR5384] (AB109497)

Immunohistochemistry (Frozen sections) analysis of mouse adrenal gland tissue sections labeling PBR with Purified ab109497 at 1/250 (3.8 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.

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Anti-PBR antibody [EPR5384] (AB109497)

TSPO (PBR) expression is localized to the mitochondria

(A) Panel shows confocal images of TSPO (red), VDAC1 (green) and nuclear counterstain (blue) in MA-10 Leydig (mouse). cells. (B) Negative control panel. Colocalization of TSPO to the mitochondrial protein VDAC1 validates the specific localization of TSPO. Scale bar 20 μm.

Cells were fixed with 4% formaldehyde and permeabilized using 0.1% Triton X-100. Cells were then blocked using 5% normal goat serum and incubated with ab109497 at 1/200 dilution.

Note : TSPO and PBR are alternative names for the same target.

Morohaku, K. et al Send to PLoS One. 2013 Sep 5;8(9):e74509. doi: 10.1371/journal.pone.0074509. eCollection 2013 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• WB

Lab

Western blot - Anti-PBR antibody [EPR5384] (AB109497)

Lanes 1- 2 : Merged signal (red and green). Green - ab109497 observed at 17 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab109497 was shown to react with PBR in wild-type HCT116 cells in western blot. Loss of signal was observed when knockout cell line ab266878 (knockout cell lysate ab257067) was used. Wild-type HCT116 and TSPO knockout HCT116 cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab109497 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 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.

All lanes:

Western blot - Anti-PBR antibody [EPR5384] (ab109497) at 1/1000 dilution

Lane 1:

Wild-type HCT116 cell lysate at 20 µg

Lane 2:

Western blot - Human TSPO (PBR) knockout HCT116 cell lysate (<a href='/en-us/products/cell-lysates/human-tspo-pbr-knockout-hct116-cell-lysate-ab257067'>ab257067</a>) at 20 µg

Predicted band size: 19 kDa

Observed band size: 17 kDa

false

• WB

Lab

Western blot - Anti-PBR antibody [EPR5384] (AB109497)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-PBR antibody [EPR5384] (ab109497) at 1/50000 dilution

Lane 1:

U-87MG cell lysate at 20 µg

Lane 2:

HEK293 cell lysate at 20 µg

Lane 3:

A431 cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 19 kDa

Observed band size: 18 kDa

false

• WB

Lab

Western blot - Anti-PBR antibody [EPR5384] (AB109497)

Lanes 1- 2 : Merged signal (red and green). Green - ab109497 observed at 15 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab109497 was shown to react with PBR in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264942 (knockout cell lysate ab257066) was used. Wild-type HeLa and TSPO knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab109497 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 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.

All lanes:

Western blot - Anti-PBR antibody [EPR5384] (ab109497) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human TSPO (PBR) knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-tspo-pbr-knockout-hela-cell-lysate-ab257066'>ab257066</a>) at 20 µg

Predicted band size: 19 kDa

Observed band size: 17 kDa

false

• WB

Lab

Western blot - Anti-PBR antibody [EPR5384] (AB109497)

Lanes 1 - 4 : Merged signal (red and green). Green - ab109497 observed at 15 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab109497 was shown to specifically react with PBR in wild type HAP1 cells. No band was observed when PBR knockout samples were used. Wild-type and PBR knockout samples were subjected to SDS-PAGE. ab109497 and ab8245 (loading control to GAPDH) were diluted at 1/10000 and 1/10000 dilution respectively and incubated overnight at 4C. 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PBR antibody [EPR5384] (ab109497) at 1/10000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

PBR knockout HAP1 cell lysate at 20 µg

Lane 3:

HEK293 cell lysate at 20 µg

Lane 4:

A431 cell lysate at 20 µg

Predicted band size: 19 kDa

false

• WB

Lab

Western blot - Anti-PBR antibody [EPR5384] (AB109497)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-PBR antibody [EPR5384] (ab109497) at 1/50000 dilution

Lane 1:

RAW264.7 cell lysate at 20 µg

Lane 2:

NIH/3T3 cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 19 kDa

Observed band size: 18 kDa

false