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Figure 1. β3-tubulin expression visualised with HB6639 used to label cultured rat neurones. HB6639 staining revealed a dense network of neuronal processes created by DIV21 cultured rat neurones. 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. HB6639 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:500 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica DM2500 epifluorescence microscope (20x objective) coupled to a Leica DFC7000T colour digital camera with DAPI LP (3.4x gain, 15.0ms exposure) and I3 (3.4x gain, 582.4ms exposure) filters. The image was processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool. |
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Figure 2. β3-tubulin expression in various tissue lysates and preparations. HB6639 revealed a single band of size 53kDa primarily present in brain cytosol fractions. Endogenous mouse IgGs were also detected by the secondary antibody in mouse tissue. Method: mouse brain and rat brain membrane (P2) and 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). Samples were loaded (20µg / lane) onto a 10% acrylamide gel alongside a protein ladder (Thermofisher, 26616) before being run at 60V for 40 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 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6639 at a 1:1000 dilution (1µg/ml). Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) 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). |
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Figure 3. The effect of varying HB6639 concentration upon staining in the stratum pyramidale of hippocampal CA1. HB6639 successfully labelled cell bodies and dendritic processes at a range of dilutions in 40µm rat hippocampal sections. Method: hippocampi were dissected from rat brains and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) for another 24hrs. A freezing microtome was used to cut 40µm transverse slices before sections were incubated in 0.05M glycine for 30 minutes. Sections were blocked in 1% BSA, 22.52mg/ml glycine before incubation overnight in varying concentrations of HB6639 (1:500 to 1:4000 dilutions, 0.25-2 µg/ml). This was followed by a two hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured using a Leica DM2500 epifluorescence microscope (20x objective) coupled to a Leica DFC7000T colour digital camera with DAPI and I3 filters. Exposure times were as follows:
1:500 – DAPI: 5.1x gain,34.6ms exposure; I3 5.1x gain, 239.5ms exposure
1:1,000 – DAPI: 5.1x gain, 24.0ms exposure; I3 5.1x gain, 123.5ms exposure
1:2,000 – DAPI: 5.1x gain, 28.8ms exposure; I3: 5.1x gain, 196.1ms exposure
1:4,000 – DAPI: 5.1x gain, 29.1ms exposure; I3: 5.1x gain, 224.3ms exposure
No primary – DAPI: 10.18ms exposure, I3: 112.ms exposure (taken using different microscope: Leica DMI6000B with Photometric-Prime95B camera).
Images were processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool before being stacked and made into a montage. |
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Figure 4. Pyramidal neurones in rat CA1 visualised using HB6639. HB6639 mediated staining of β3-tubulin successfully labelled pyramidal neurones in the CA1 of rat hippocampi at a dilution of 1:500 (2µg/ml). Method: hippocampi were dissected from rat brains and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) for another 24hrs. A freezing microtome was used to cut 40µm transverse slices before sections were incubated in 0.05M glycine for 30 minutes. Sections were blocked in 1% BSA, 22.52mg/ml glycine before incubation overnight in HB6639 (1:500 dilution, 2 µg/ml). This was followed by a two hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. The image was captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. The image was captured using a 20x objective and 405 (27% power) / 488 (42% power) laser lines. The image was processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682). |
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Figure 5. β3-tubulin expression in various tissue lysates and preparations with GAPDH loading control. HB6639 revealed a single band of size 51kDa primarily present in brain cytosol fractions. Method: mouse brain and rat brain membrane (P2) and 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). 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 30 minutes followed by 120V for 100 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6639 at a 1:1000 dilution (1µg/ml) and HB9177 (anti-GAPDH monoclonal antibody) at a 1:2,000 dilution (0.5µg/ml). Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) 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). |
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Figure 6. Concentration response of HB6639 staining in a rat brain cytosol preparation. HB6639 shows consistent results with low background at dilutions as low as 1:8,000 (125 ng/ml). Method: cytosol fractions were prepared from fresh rat brains following established protocols (Molnar et al., 1993. Neuroscience 53:307-326). Rat cytosol samples were loaded (20µg / lane) onto a 10% acrylamide gel alongside a protein ladder (Thermofisher, 26616) before being run at 60V for 41 minutes followed by 120V for 85 minutes. Wet transfer to a PVDF membrane was completed in 93 minutes using 400mA. Following transfer the membrane was cut into strips between markers. Strips were blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6639. Each strip was incubated separately with a separate HB6639 concentration with this ranging from 2µg/ml (1:500 dilution) to 125ng/ml (1:8,000 dilution). Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) 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). Band intensity was calculated using Image Studio version 5.2.5 (LiCor) and a graph was constructed in GraphPad Prism 9 using a 3-parameter Hill equation curve fit. |
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Figure 7. β3-tubulin expression visualised with HB6639 used to label cultured rat neurones. HB6639 staining revealed a dense network of neuronal processes created by DIV21 cultured rat neurones. 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. HB6639 was incubated overnight (4°C) at a 1:500 dilution (2µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. 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 using a 40x objective (1.28x zoom), 405nm (43.5% power, gain: 658) and 488nm (44.6% power, gain: 998) laser lines in a z-stack (0.24 µm spacing). Deconvolution was carried out using Huygens Essential (Scientific Volume Imagine) followed by the stack being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682). |
