Anti-NeuN antibody ValidAb™

HB6498 stains the NeuN expressing neurones of rat CA1 while HB6457 (mouse monoclonal anti-parvalbumin) stains the subset expressing parvalbumin. 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 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6498 (1:5000 dilution, 0.2µg/ml) and HB6457 (1:1000 dilution, 1µg/ml). This was followed by a two hour incubation with polyclonal goat anti-mouse DyLight 488 conjugated (Thermofisher 35503, 1:300 dilution) and polyclonal goat anti-rabbit DyLight 594 (Thermofisher 35561) 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 a tilescan using a Leica DMI6000B inverted epifluorescence microscope coupled to a Photometric Prime 95B camera utilising a 20x objective and A4 (100ms exposure @ 1.7x gain) / GFP (161ms exposure @3x gain) / TX2 (410ms @ 4.6x gain) filters. Images were captured as a stack (3.4µm 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).

HB6498 stains the NeuN expressing neurones found within the rat hippocampal formation. 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 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6498 (1:2000 dilution, 0.5µg/ml). This was followed by a two hour incubation with a polyclonal goat anti-rabbit DyLight 594 (Thermofisher 35561) secondary antibody. 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 a tilescan using a Leica DMI6000B inverted epifluorescence microscope coupled to a Photometric Prime 95B camera utilising a 10x objective and DAPI (26ms exposure @ 5.8x gain) / Y3 (177ms exposure @ 5.4x gain) filters. Images were captured as a stack (5.4µm 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).

HB6498 conjugated to Janelia Fluor 525 (HB8455) stains the NeuN expressing neurons of the dentate gyrus. Method: HB6498 was conjugated to Janelia Fluor 525 SE (for protocol see HB8455). Rat brains were then 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 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6498 (4µg/ml) DAPI (HB0747) was then incubated at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. The image was captured using a Leica DMI6000B inverted epifluorescence microscope coupled to a Photometric Prime 95B camera utilising a 10x objective and DAPI (17ms exposure) / TX2 (200ms 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).

HB6498 stains NeuN expression neurones in the hippocampus at concentrations as low as 62.5ng/ml (1 in 16,000 dilution). 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 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in dilutions of HB6498 ranging from 1 in2000 (500ng/ml) to 1 in 16,000 (62.5ng/ml). This was followed by a two hour incubation with a polyclonal goat anti-rabbit DyLight 594 (Thermofisher 35561) secondary antibody. 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 DMI6000B inverted epifluorescence microscope coupled to a Photometric Prime 95B camera utilising a 10x objective and DAPI/TX2 filters. Exposure times were:

• 1 in 2,000 dilution: DAPI: 314ms, TX2: 113ms
• 1 in 4,000 dilution: DAPI: 314ms, TX2: 240ms
• 1 in 8,000 dilution: DAPI: 314ms, TX2: 240ms
• 1 in 16,000 dilution: DAPI: 314ms, TX2: 240ms

Images were captured as a stack (6.1µm 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).

HB6498 revealed the 76kDa (Synapsin I), 50kDa (FOX3) and 45kDa (FOX3) bands associated with the NeuN antigen. 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 100V for 80 minutes then 130V for 40 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 HB6498 at a 1:1,000 dilution (1µg/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 with two changes of stripping buffer (HB7756) before being washed, blocked for 2 hours in 5% non-fat dry milk and incubated in HB9177 (mouse monoclonal anti-GAPDH, 1:4,000 dilution, 0.25µg/ml) and HB8224 (rabbit polyclonal anti-ß-actin, 1:1000 dilution) overnight at 4°C. Following washing the membrane was incubated in secondary antibodies (1:10,000 dilution for both, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682 and Polyclonal Goat anti-rabbit HRP conjugated, Sigma Aldrich A6154) for 2hrs and visualised again using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure).

HB6498 shows strong affinity for NeuN with bands visible at as low as 0.031µg/ml (1 in 32,000 dilution). At very high concentrations an additional band at ≈27kDa is visible which is not stained at dilutions below 1in2000. 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 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. 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 HB6498. Each strip was incubated separately with a separate HB6498 concentration with this ranging from 4µg/ml (1:250 dilution) to 0.031µg/ml (1:32,000 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.

HB6429 (mouse monoclonal anti-NeuN) and HB6498 (rabbit polyclonal anti-NeuN) show overlapping patterns of staining in rat cortex providing strong evidence for 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 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6498 (1:5000 dilution, 0.2µg/ml) and HB6429 (1:1000 dilution, 1µg/ml). This was followed by a two hour incubation with polyclonal goat anti-mouse DyLight 488 conjugated (Thermofisher 35503, 1:300 dilution) and polyclonal goat anti-rabbit DyLight 594 (Thermofisher 35561) 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 using a Leica DMI6000B inverted epifluorescence microscope coupled to a Photometric Prime 95B camera utilising a 20x objective and DAP (10ms exposure) / L5 (435ms exposure) / TX2 (118ms exposure) filters. Images were captured as a stack 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).

HB6498 conjugated to Janelia Fluor 525 (HB8455) stains NeuN expressing neurons in the rat brain. Method: HB6498 was conjugated to Janelia Fluor 525 SE (for protocol see HB8455). Rat brains were then 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 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6498 (4µg/ml) DAPI (HB0747) was then incubated at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. The image was captured using a Leica DMI6000B inverted epifluorescence microscope coupled to a Photometric Prime 95B camera utilising a 20x objective and DAPI (8ms exposure) / TX2 (150ms exposure) filters. Images were captured as a stack (0.6µm 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).

HB6498 stains the NeuN expressing neurones found within the rat hippocampal formation. 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 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6498 (1:2000 dilution, 0.5µg/ml). This was followed by a two hour incubation with a polyclonal goat anti-rabbit DyLight 594 (Thermofisher 35561) secondary antibody. 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 DMI6000B inverted epifluorescence microscope coupled to a Photometric Prime 95B camera utilising a 10x objective and DAPI (314ms exposure) / TX2 (113ms 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).