Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547)

Immunostaining of formalin fixed paraffin embedded human microtissues after exposure to MTX, TAA and TGF-β1.

Formalin fixed paraffin embedded slides of HepaRG/THP-1 macrophages/hTERT-HSC microtissues were stained with Hematoxylin & Eosin (H&E) and vimentin after 14 days of treatment with MTX, TAA and TGF-β1. Microtissues were fixed in 4% PFA and embedded in 2% agarose prior to paraffinization. Microtissues showed increase in the vimentin positive cells after MTX, TAA and TGF-β1 exposure. Vimentin stainings show proliferation of stellate cells and THP-1 macrophages in the microtissues, suggesting the onset of inflammation process.

For full image see PMID 28665955.

Image from Prestigiacomo V et al. PLoS One. 2017;12(6):e0179995. Fig 7.; doi: 10.1371/journal.pone.0179995.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547)

Immunohistochemical staining of paraffin embedded paraformaldehyde fixed rhesus monkey retina tissue with ab92547 (green) at a working dilution of 1/200. The sample was incubaded with the primary antibody fro 20 hours, at 4°C in 2.5% serum. The secondary antibody used is a Goat anti-rabbit AlexaFluor 488 at 1/400. Heat mediated antigen retrieval was perfomed using citrate pH 6. Tissue was blocked with 5% serum for 1 hour 30 minutes at 25°C

Western blot - Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using LI-COR® blocking buffer before being incubated with ab92547 overnight at 4°C. Antibody binding was detected using the IRDye® 800CW Goat Anti-Rabbit secondary at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Odyssey® CLx Imaging System.

All lanes: Western blot - Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547) at 1/1000 dilution

Lane 1: HeLa (Human epithelial cell line from cervix adenocarcinoma) Whole Cell Lysate at 20 µg

Lane 2: HEK293 (Human epithelial cell line from embryonic kidney) Whole Cell Lysate at 20 µg

Lane 3: Jurkat (Human T cell lymphoblast-like cell line) Whole cell lysate at 20 µg

Lane 4: A549 (Human lung carcinoma cell line) Whole cell lysate at 20 µg

Lane 5: NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 20 µg

Lane 6: PC12 (Rat adrenal gland pheochromocytoma cell line) Whole Cell Lysate at 20 µg

Lane 7: HUVEC (Human umbilical vein endothelial cell line) Whole cell lysate at 20 µg

Lane 8: A431 (Human epidermoid carcinoma cell line) Whole cell lysate at 20 µg

Lane 9: Daudi (Human Burkitt's lymphoma cell line) Whole cell lysate at 20 µg

Lane 10: Caco 2 (Human colorectal adenocarcinoma cell line) Whole Cell Lysate at 20 µg

Secondary

All lanes: IRDye® 800CW Goat Anti-Rabbit at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 53 kDa

Observed band size: 53 kDa

Western blot - Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547)

Different batches of ab92547 were tested on HEK-293 (Human embryonic kidney epithelial cell) lysate at 0.02 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 54 kDa.

All lanes: Western blot - Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547)

Predicted band size: 53 kDa

Flow Cytometry (Intracellular) - Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547)

Overlay histogram showing HAP1 wildtype (green line) and HAP1-VIM knockout cells (red line) stained with ab92547. The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab92547, 0.5μg/ml) for 30 min at 22°C. The secondary antibody used wasGoat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibodyat 1/2000 dilution for 30 min at 22°C. A Rabbit IgG isotype control antibody (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-VIM knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity). Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.