Recombinant Anti-PDGFR alpha antibody [EPR22059-270] (ab203491)

Immunohistochemical analysis of paraffin-embedded human endometrium tissue labeling PDGFR alpha with ab203491 at 0.26 µg/mL followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in human endometrium was observed. The section was incubated with ab203491 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used.

Lanes 1 - 2: Merged signal (red and green). Green - ab203491 observed at 150 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab203491 was shown to react with PDGFR alpha in wild-type SH-SY5Y cells in Western blot with loss of signal observed in PDGFRA knockout cell line ab275335 (knockout cell lysate ab275522). Wild-type SH-SY5Y and PDGFRA knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab203491 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

ab203491 staining PDGFR alpha in wild-type SH-SY5Y cells (top panel) and PDGFRA knockout SH-SY5Y cells (ab275335) (bottom panel). The cells were fixed with 100% methanol (5 min) then 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 ab203491 at 1μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution 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) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A-204 (human muscle rhabdomyosarcoma cell line) cells and A-172 (human brain glioblastoma cell line) labeling PDGFR alpha with ab203491 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing membranous and cytoplasmic staining in A-204 cell line.

Negative control: A-172 (PMID:8425771.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time : Lane 1: 3 minutes; Lane 2: 59 seconds.

Immunohistochemical analysis of paraffin-embedded mouse uterus tissue labeling PDGFR alpha with ab203491 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Positive staining on interstitial cells of mouse uterus (PMID: 25788664). Counter stained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. 

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

PDGFR alpha was immunoprecipitated from 0.35 mg of A-204 (human muscle rhabdomyosarcoma cell line) whole cell lysate with ab203491 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab203491 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1: A-204 whole cell lysate 10 µg (Input). 

Lane 2: ab203491 IP in A-204 whole cell lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab203491 in A-204 whole cell lysate.

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

Exposure time: 50 seconds.

Flow cytometric analysis of NIH/3T3 (mouse embyro fibroblast cell line) cell line labeling PDGFR alpha with ab203491 at 1/500 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

Gated on viable cells.

IHC image of PDGFR alpha staining in a section of frozen normal human kidney performed on a Leica BOND^TM system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab203491, 1/500 dilution, 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.

Immunohistochemical analysis of paraffin-embedded rat E14.5 intervertebral disc tissue labeling PDGFR alpha with ab203491 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Positive staining in mesenchymal cells of rat E14.5 intervertebral disc (PMID: 9199674). Counter stained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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

Immunohistochemical analysis of paraffin-embedded mouse E14.5 lung tissue labeling PDGFR alpha with ab203491 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Positive staining in the mesenchyme of mouse E14.5 lung (PMID: 8681381). Counter stained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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

Immunohistochemical analysis of paraffin-embedded mouse E14.5 intervertebral disc tissue labeling PDGFR alpha with ab203491 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Positive staining in mesenchymal cells of mouse E14.5 intervertebral disc (PMID: 9199674). Counter stained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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

Blocking/Dilution buffer: 5% NFDM/TBST.

Flow cytometric analysis of A-172 (human brain glioblastoma cell line, left) and A-204 (human muscle rhabdomyosarcoma cell line, right) cell lines labeling PDGFR alpha with ab203491 at 1/50 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

Gated on viable cells.