Anti-Vimentin antibody ValidAb^TM

Glia in the CNS have a mesenchymal origin therefore co-express vimentin (labelled with HB7843) and GFAP (labelled with HB8267). Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 before being blocked in 1% BSA, 300mM glycine. Cells were incubated overnight at 4°C in HB7843 (2µg/ml, 1:500) and HB8267 (1µg/ml, 1:1000). Secondary antibodies were incubated for 1 hour at RT (donkey polyclonal anti-mouse Dy488, Thermofisher SA5-10166, 1:300 and donkey polyclonal anti-goat Dy594, Thermofisher SA5-10088, 1:300) before nuclei were counterstained using DAPI (HB0747, 1µg/ml). For more detail please see our ICC protocol. Images were captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. The image was captured in Lightning deconvolution mode using a 63x objective with 0.30µm z-spacing, 405nm (11.8% power @ 851V gain (PMT)), 496nm (0.5% power @ 17.7% gain (Hyd)) and 561nm (2.0% power @ 14.7% gain (HyD)) lasers. Following capture the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Both HB6497 (mouse monoclonal) and HB7843 (goat polyclonal) immunoreactivity within mixed neuronal cultures overlaps therefore providing evidence for antibody specificity. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 before being blocked in 1% BSA, 300mM glycine. Cells were incubated overnight at 4°C in HB6497 (1µg/ml, 1:1000) and HB7843 (2µg/ml, 1:500). Secondary antibodies were incubated for 1 hour at RT (donkey polyclonal anti-mouse Dy488, Thermofisher SA5-10166, 1:300 and donkey polyclonal anti-goat Dy594, Thermofisher SA5-10088, 1:300) before nuclei were counterstained using DAPI (HB0747, 1µg/ml). For more detail please see our ICC protocol. Images were captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. The image was captured in Lightning deconvolution mode using a 63x objective with 0.27µm z-spacing, 405nm (11.8% power @ 825V gain (PMT)), 561nm (1.0% power @ 10.8% gain (Hyd)) and 496nm (0.5% power @ 27.1% gain (HyD)) lasers. Following capture the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Both HB6497 (mouse monoclonal) and HB7843 (goat polyclonal) immunoreactivity within mixed neuronal cultures overlaps therefore providing evidence for antibody specificity. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 before being blocked in 1% BSA, 300mM glycine. Cells were incubated overnight at 4°C in HB6497 (1µg/ml, 1:1000) and HB7843 (2µg/ml, 1:500). Secondary antibodies were incubated for 1 hour at RT (donkey polyclonal anti-mouse Dy488, Thermofisher SA5-10166, 1:300 and donkey polyclonal anti-goat Dy594, Thermofisher SA5-10088, 1:300) before nuclei were counterstained using DAPI (HB0747, 1µg/ml). For more detail please see our ICC protocol. Images were captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. The image was captured in Lightning deconvolution mode using a 63x objective with 0.27µm z-spacing, 405nm (11.8% power @ 825V gain (PMT)), 561nm (1.0% power @ 10.8% gain (Hyd)) and 496nm (0.5% power @ 27.1% gain (HyD)) lasers. Following capture the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Vimentin and GFAP are both expressed in glial cells and can be seen to express at different levels in individual cells creating a colour gradient across the cell population. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB8267 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) with HB7843 being incubated at a 1:500 dilution (2µg/ml). Secondary antibodies (Polyclonal donkey anti-mouse DyLight 488 conjugated, Thermofisher SA5-10166, 1:300 dilution and Polyclonal donkey anti-goat DyLight 594 conjugated, Thermofisher SA5-10088, 1:300 dilution) were incubated for 1 hour at room temperature. Nuclei were visualised using 1µg/ml DAPI (HB0747). For more detail please see our ICC protocol. Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured in a tile scan and z-stack (0.49µm spacing) using Lightning adaptive deconvolution and a 63x objective. Lasers used were 405nm (20.0% power, PMT: 712.7V gain), 496nm (1.0% power, HyD: 14.1% gain) and 561nm (1.5% power, HyD: 10% gain). The tile scan was merged using LASX before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

HB7843 optimally reveals the dense network of vimentin containing intermediate filaments at 2µg/ml (1:500 dilution). Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 before being blocked in 1% BSA, 300mM glycine. Cells were incubated overnight at 4°C in HB7843 (concentrations ranging from 2µg/ml(1:500) to 0.25µg/ml (1:4000)) before being incubated in secondary antibody (donkey polyclonal anti-goat Dy594, Thermofisher SA5-10088, 1:300) for 1 hour at RT. Nuclei were counterstained using DAPI (HB0747, 1µg/ml). For more detail please see our ICC protocol. Images were captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. Images were captured in Lightning deconvolution mode using a 63x objective with the following exposure settings:

• 1:500, 405nm: 11.8% power, 851V gain (PMT); 496nm: 0.5% power, 17.7% gain (HyD); 561nm: 2.0% power, 14.7% gain (HyD),
• 1:1000, 405nm: 11.8% power, 851V gain (PMT); 496nm: 0.5% power, 24.9% gain (HyD); 561nm: 2.0% power, 26.7% gain (HyD),
• 1:2000, 405nm: 11.8% power, 851V gain (PMT); 496nm: 1.0% power, 32.0% gain (HyD); 561nm: 5.0% power, 22.1% gain (HyD),
• 1:4000, 405nm: 11.8% power, 851V gain (PMT); 496nm: 1.0% power, 10.0% gain (HyD); 561nm: 5.0% power, 42.9% gain (HyD),

Following capture the stacks were flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

HB7843 reveals the network of intermediate filaments present within cultured HeLa cells. Method: HeLa cells were cultured in 10% FBS, 1% pen/strep in DMEM before being plated onto PDL coated coverslips and incubated for 2 days. Cells were then fixed in 4% PFA before being permeabilised with 0.1% Triton X-100. Cells were blocked in 1% BSA, 300mM glycine then HB7843 was incubated at 4°C overnight (1:500, 2µg/ml). Following washing, cells were incubated in a polyclonal donkey anti-goat DyLight 594 conjugated secondary antibody (1:300, Thermofisher SA5-10088) for 1 hour at room temperature. Nuclei were stained using 1µg/ml DAPI (HB0747). For more detail please see our ICC protocol. The image was captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured in a z-stack (0.37µm spacing) using Lightning adaptive deconvolution and a 63x objective. Lasers used were 405nm (3.2% power, PMT: 542.1V gain) and 561nm (1.0% power, HyD: 28.6% gain). Following capture, the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

HB7843 revealed a band corresponding with Vimentin at 55.6kDa in human cell line samples and brain cytosol preparations from mouse and rat. There was much immunoreactivity in liver samples. Method: mouse brain and rat brain cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Other tissue lysates were prepared following established protocols from freshly dissected tissue (see our guide on WB sample preparation). HEK293 and HeLa cells were cultured in 10% FBS, 1% pen/strep in DMEM before having proteins extracted. Samples were loaded (20µg / lane) onto a 12% acrylamide gel alongside a protein ladder (Biorad precision plus dual colour, 1610374) before being run at 60V for 45 minutes followed by 120V for 90 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 1hr in 5% non-fat dry milk before being incubated overnight at 4°C in HB7843 at a 1:1,000 dilution (1µg/ml). Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal donkey anti-goat HRP conjugated, Thermofisher 77-56-060520) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure). Following imaging, the membrane was stripped with two changes of stripping buffer (HB7756) before being washed, blocked for 30 minutes in 1% fish skin gelatin and incubated in HB9177 (1:4,000 dilution, 0.25µg/ml) for 3 days at 4°C. Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 2hrs and visualised again using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 2 min exposure, 700nm channel: 30 sec exposure).