Anti-Tyrosine hydroxylase antibody ValidAb™

HB6605 staining specifically labelled the dopaminergic neurones present in mouse striatum and nucleus accumbens. Image shown is from bregma ≈ 1.145mm. Abbreviations: CTX cortex, cc corpus callosum, SN septal nucleus, VL lateral ventricle, CP caudate putamen, aco anterior commissure, Nacc nucleus accumbens, OT olfactory tubercle. Method: Mice were perfused with PBS then 4% PFA before brains were removed and 40µm coronal sections prepared using a freezing microtome. Sections were incubated in 1% NaBH4 for 30 minutes then 0.05M glycine for 30 minutes. Section were blocked in 3% goat serum, 2% BSA for 2hrs before incubation overnight in HB6605 (1:1000 dilution, 150ng/ml) at 4°C. Sections were incubated with secondary antibody (Polyclonal goat anti-rabbit DyLight 488 conjugated, Thermofisher 35552, 1:150 dilution). For more detail please see our IHC(IF) protocol . Images were captured using a Leica DMI6000B inverted epifluorescence microscope (20x objective) coupled to a Leica DFC365FX monochrome digital camera with a GFP (89.1ms exposure and 3.2x gain) filter. Acquisition was carried out as both a z-stack (3.44µm stack spacing) and a tile scan which was merged using the mosaic merge function in LASX. The image was further processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using a Z-projection (maximum intensity) and using the subtract background (50px rolling ball radius) tool.

HB6429 labels the nuclei of cell bodies in the striatum whereas HB6605 labels dopaminergic projections. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm transverse slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6429 (1:1000 dilution, 1µg/ml) and HB6605 (1:1,000 dilution). This was followed by a two hour incubation with polyclonal donkey anti-mouse DyLight 488 conjugated (Thermofisher SA5-10166, 1:300 dilution) and polyclonal goat anti-rabbit Janelia Fluor 525 conjugated secondary antibodies. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. The image was captured as using a Leica DMI6000B inverted epifluorescence microscope coupled to a Photometric Prime 95B camera utilising a 10x objective and DAPI (8ms exposure) / L5 (200ms exposure) / RHO (250ms exposure) filters. Images were captured as a stack (4.3µm z-spacing) before being deconvolved using Huygens Professional software then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

HB6605 labels the dense network of dopaminergic fibres in the caudate putamen whereas HB8014 labels myelin containing axons. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 15 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6605 (1:2000) and HB8014 (1:2,000 dilution). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse DyLight 488, Thermofisher, 35503 and goat anti-rabbit DyLight 650, Thermofisher 11804574). Propidium Iodide was included in the mounting media to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured using Leica DMI6000B inverted epifluorescence microscope attached to a DFC365FX monochrome digital camera. The image was captured as a tilescan and z-stack (2.4µm spacing) using a 20x objective, GFP (583.7ms @ 5.5x gain), Y3 (210ms @ 4.4x gain) and Y5 (742ms @ 5.2x gain). The image was merged in LASX then deconvolved in Huygens professional before being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

The staining pattens of HB6605 and HB6589 overlap in rat caudate putamen (CPu) showing strong evidence for antibody specificity. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 15 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6589 (1:5,000 dilution), HB8014 (1:1,000 dilution) and HB6605 (1:4,000 dilution). This was followed by a two-hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken DyLight 488, Thermofisher, A78948, polyclonal goat anti-mouse DyLight 550, Thermofisher, 84540 and polyclonal goat anti-rabbit DyLight 650, Thermofisher, 11804574). Sections were mounted using MightyMount Antifade Fluorescence Mounting Medium with DAPI (aqueous). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. The image was captured using a 10x objective in a z-stack (5µm spacing). The image was captured as a tilescan using DAP (38ms exposure), L5 (264ms exposure), TX2 (61.8ms exposure) and Y5 (500ms exposure) filters. The stack was deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

The striatum is a key projection site for dopaminergic neurones. HB6605 stains the dense network of dopaminergic terminals found within the striatum in rat brain sections. Method: Brains were dissected from adult rats and fixed for 48hrs in 4% PFA before then incubated in 30% sucrose (in PBS) until the brains had sunk. A freezing microtome was used to cut 40µm coronal slices before sections were incubated in 1% NaBH4 for 30 minutes then 0.05M glycine for 30 minutes. Sections were blocked in 2% BSA, 3% goat serum before incubation overnight in HB6605 (1:1000, 150ng/ml) at 4°C. This was followed by a two hour incubation with secondary antibody (Polyclonal goat anti-rabbit DyLight 488 conjugated, Thermofisher 35552, 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 SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. The image was captured using a 40x objective (1.5x zoom), 405nm (24.5% power, PMT: 509V) and 488nm (30.1% power, PMT: 629V) laser lines in a z-stack (0.43µm 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).

HB6433 revealed primary bands at 59kDa and 63kDa corresponding to unphosphorylated and phosphorylated forms of the protein. There was also a 50kDa band present in rat liver which could not be identified and a 33kDa band in the rat brain P2 membrane fraction which is believed to be a degradation product. 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 40 minutes followed by 130V 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 HB6605 at a 1:1000 dilution (150ng/ml). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-rabbit HRP conjugated, Sigma Aldrich A6154) 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 by incubation with two changes of stripping buffer (HB7756) then was re-probed following the protocol described with HB9177 (mouse monoclonal anti-GAPDH) at a 1:4000 dilution (250ng/ml) overnight 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: 4 min exposure, 700nm channel: 30 sec exposure).

HB6605 generated specific staining of dopaminergic terminals in the striatum at dilutions down to 1:2000 (75ng/ml). Method: Brains were dissected from adult rats and fixed for 48hrs in 4% PFA before then incubated in 30% sucrose (in PBS) until the brains had sunk. A freezing microtome was used to cut 40µm coronal slices before sections were incubated in 1% NaBH4 for 30 minutes then 0.05M glycine for 30 minutes. Sections were blocked in 2% BSA, 3% goat serum before incubation overnight in HB6605 ranging in concentration from 150ng/ml to 9.4ng/ml (1:1000 to 1:16,000 dilutions) at 4°C. This was followed by a two hour incubation with secondary antibody (Polyclonal goat anti-rabbit DyLight 488 conjugated, Thermofisher 35552, 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 DMi8 inverted epifluorescence microscope (40x objective) coupled to a Leica DFC365FX monochrome digital camera with DAPI LP and FITC LP filters. Exposure times were as follows: 

• 1:1,000 – DAPI LP: 16.8ms @ 1x gain; FITC LP: 322.1ms @ 1x gain
• 1:2,000 – DAPI LP: 16.8ms @ 1x gain; FITC LP: 322.1ms @ 1.7x gain
• 1:4,000 – DAPI LP: 35.5ms @ 1x gain; FITC LP: 180.9ms @ 2.5x gain
• 1:8,000 – DAPI LP: 23.5ms @ 1x gain; FITC LP: 180.9ms @ 2.5x gain
• 1:16,000 – DAPI LP: 23.5ms @ 1x gain; FITC LP: 336.4ms @ 3.2x gain

Images were captured as a z-stack and were processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using a maximal Z-projection then the subtract background (50px rolling ball radius) tool followed by stacking and montage creation. 

HB6605 works down to a 1:2000 (75ng/ml) dilution in Western blot experiments. 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 (BioRad Precision Plus dual colour, 1610374) before being run at 65V for 30 minutes followed by 180V for 60 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. Following transfer the membrane was cut into strips using Ponceau dye to visualise and cut individual lanes. Strips were blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6605. Each strip was incubated separately with a separate HB6605 concentration with this ranging from 300ng/ml (1:500 dilution) to 1.2ng/ml (1:128,000 dilution). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-rabbit HRP conjugated, Sigma Aldrich A6154) 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.

The striatum is a key projection site for dopaminergic neurones. HB6605 stains the dense network of dopaminergic terminals found within the striatum in rat brain sections. Method: Brains were dissected from adult rats and fixed for 48hrs in 4% PFA before then incubated in 30% sucrose (in PBS) until the brains had sunk. A freezing microtome was used to cut 40µm coronal slices before sections were incubated in 1% NaBH4 for 30 minutes then 0.05M glycine for 30 minutes. Sections were blocked in 2% BSA, 3% goat serum before incubation overnight in HB6605 (1:2000, 75ng/ml) at 4°C. This was followed by a two hour incubation with secondary antibody (Polyclonal goat anti-rabbit DyLight 488 conjugated, Thermofisher 35552, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol 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 (1.5x zoom), 405nm (24.5% power, PMT: 509V) and 488nm (30.1% power, PMT: 629V) laser lines in a z-stack (0.43µm 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).