Anti-TGF beta 1 抗体 [EPR21143] (ab215715)

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Immunohistochemical analysis of formalin-fixed paraffin-embedded human bone labelling TGF beta 1 with ab215715 at a concentration of 3µg/ml. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with a Bond™ Polymer Refine Detection kit. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20 mins. ab215715 anti-TGF beta 1 antibody [EPR21143] was incubated for 15mins at room temperature. Sections were counterstained with Hematoxylin. Image inset shows absence of staining in secondary antibody only control.

Immunohistochemical analysis of formalin-fixed paraffin-embedded human bone labelling TGF beta 1 with ab215715 at a dilution of 4µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32min with ULTRA cell conditioning solution (CC1 pH8.5). ab215715 anti TGF beta 1 antibody was incubated at 37°C for 16min. Sections were counterstained is with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

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

ab181602 was used as a GAPDH loading control.

For testing samples with low expression level of TGF beta 1, we recommend ab179695 which could give stronger signal.  Loading larger amount of lysate or lower antibody dilution would also help.

Lanes 1 - 4: Merged signal (red and green). Green - ab215715 observed at 13 and 44 kDa. Red - loading control, ab7291, observed at 50 kDa.

ab215715 was shown to specifically react with in wild-type HeLa cells as signal was lost in TGFB1 knockout cells. Wild-type and TGFB1 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% NF Milk. Ab215715 and ab7291 (Mouse anti-tubulin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

Lanes 1 - 4: Merged signal (red and green). Green - ab215715 observed at 48 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab215715 was shown to react with TGF beta 1 in wild-type A549 cells in Western blot with loss of signal observed in TGFB1 knockout cell line ab269509 (TGFB1 knockout cell lysate ab269671). Wild-type A549 and TGFB1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab215715 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) 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.

Immunohistochemical analysis of paraffin-embedded human thrombocytosis tissue labeling TGF beta 1 with ab215715 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic staining of megakaryocytes in human thrombocytosis (PMID: 25305163). 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 rat spleen tissue labeling TGF beta 1 with ab215715 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining of megakaryocytes and platelets in rat spleen (PMID: 25305163). 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 spleen tissue labeling TGF beta 1 with ab215715 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining of megakaryocytes and platelets in mouse spleen (PMID: 25305163). 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.

Exposure times: Lanes 1-4: 3 minutes; Lane 5: 41 seconds; Lane 6: 24 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

The molecular mass observed is consistent with the literature (PMID 2139036).

The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument.