Fibroblast Marker (Vimentin, alpha smooth muscle Actin, Hsp47, S100A4) Antibody Panel - Human, Mouse (ab254015)

Immunohistochemical staining of paraffin embedded human cervical carcinoma with purified ab92547 at a working dilution of 1/250. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunohistochemical staining of paraffin embedded mouse kidney with purified ab92547 at a working dilution of 1/250. The secondary antibody used is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

ab92547 staining Vimentin in wild-type HAP1 cells (top panel) and VIM 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 ab92547 at 0.5μg/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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody 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).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human smooth muscle tissue labeling alpha smooth muscle Actin with purified ab32575 at a dilution of 1/200. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051, an 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.

Immunocytochemistry/Immunofluorescence analysis of A431 (human epidermoid carcinoma) cells labeling alpha smooth muscle Actin (green) with purified ab32575 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Cells were counterstained with ab7291, anti-Tubulin (mouse mAb) at 1/1000 followed by ab150120 Alexa Fluor^®594 goat anti-mouse secondary (1/1000). Nuclei were counterstained with DAPI (blue).

For negative control 1, rabbit primary antibody and anti-mouse secondary antibody (ab150120) were used. For negative control 2, ab7291 (mouse primary antibody) was used followed by anti-rabbit secondary antibody (ab150077).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse smooth muscle tissue labeling alpha smooth muscle Actin with purified ab32575 at a dilution of 1/200. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051, an 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.

Immunocytochemistry/ Immunofluorescence analysis of NIH/3T3(Mouse embryonic fibroblast) cells labeling alpha smooth muscle Actin with purified ab32575 at 1/500 dilution (5.2 µg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 dilution (2 µg/ml) dilution. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunohistochemical analysis of paraffin-embedded Human gastric carcinoma tissue labeling S100A4 using ab197896 at 1/2000 dilution. Goat Anti-Rabbit IgG H&L (HRP) ab97051 was used as a secondary antibody at 1/500 dilution. Cells were counterstained with Hematoxylin. 

Inset image: negative control obtained using PBS instead of ab197896 and secondary antibody only.
Note: Cytoplasm and nuclear staining on human gastric carcinoma tissue was observed.

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: Hsp47 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: HepG2 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - Anti-Hsp47 antibody [EPR4217] (ab109117) observed at 46 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab109117 was shown to recognize Hsp47 in wild-type HAP1 cells as signal was lost at the expected MW in Hsp47 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and Hsp47 knockout samples were subjected to SDS-PAGE. ab109117 and ab9484 (Mouse anti-GAPDH 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.

ab109117 staining Hsp47 in Human colon tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed and paraffin-embedded, antigen retrieval was by heat mediation in Tris/EDTA buffer pH9. Samples were incubated with primary antibody (1/300). An undiluted HRP-conjugated mouse anti-rabbit IgG was used as the secondary antibody. Tissue counterstained with Hematoxylin.