Goat Anti-Mouse IgG H&L (Janelia Fluor® 646) preadsorbed ValidAb™

HEK293T cells stained with for Vimentin (HB6497) using HB7393 goat anti-mouse IgG H&L (Janelia Fluor® 646) preadsorbed as the secondary antibody. Method: HEK293 cells were cultured following established protocols (Lee et al., 2019. PLoS ONE, 14(5):e0213116) before being fixed with 4% PFA. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB6497 (mouse monoclonal anti-vimentin) was incubated overnight (4°C) at a 1:2,000 dilution (0.5µg/ml) followed by a one hour incubation with HB7393 goat anti-mouse IgG H&L (Janelia Fluor® 646) preadsorbed secondary antibody (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 in Lightning deconvolution mode using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope with a 63x objective, 405nm (9.95% power, HyD: 10% gain) and 633nm (1.5% power, HyD: 10% gain) excitation lasers. Images were captured as a z-stack (0.3µm spacing) before being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Rat cerebellum stained for β3-tubulin (HB6639) using HB7393 goat anti-mouse IgG H&L (Janelia Fluor® 646) preadsorbed secondary antibody. 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 HB6639 (1:1,000 dilution, 1µg/ml). This was followed by a two-hour incubation with HB7393 goat anti-mouse IgG H&L (Janelia Fluor® 646) preadsorbed secondary antibody at a 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 as a z-stack (0.3µm spacing) using a Leica DMI6000B fluorescence microscope with a 40x objective using DAP (5ms exposure) and Y5 (100ms exposure) filters. Following capture, deconvolution was carried out using Huygens Professional before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

HEK293T cells stained with for Vimentin (HB6497) using HB7393 goat anti-mouse IgG H&L (Janelia Fluor® 646) preadsorbed as the secondary antibody. Method: HEK293 cells were cultured following established protocols (Lee et al., 2019. PLoS ONE, 14(5):e0213116) before being fixed with 4% PFA. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB6497 (mouse monoclonal anti-vimentin) was incubated overnight (4°C) at a 1:2,000 dilution (0.5µg/ml) followed by a one hour incubation with HB7393 goat anti-mouse IgG H&L (Janelia Fluor® 646) preadsorbed secondary antibody (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 in Lightning deconvolution mode using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope with a 63x objective, 405nm (9.95% power, HyD: 10% gain) and 633nm (2.8% power, HyD: 10% gain) excitation lasers. Images were captured as a z-stack (0.3µm spacing) before being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Janelia Fluor® 646 conjugated secondary antibodies show greater resistance to photobleaching alongside equivalent brightness than DyLight 650 conjugated secondaries in a HEK293T based cell assay. Method: HEK293T cells were cultured on 10mm coverslips and stained with an anti-MFF antibody (1:1,000 dilution) and either a goat anti-rabbit DyLight 650 or goat anti-rabbit Janelia Fluor® 646 conjugated secondary antibody. For more information please see our ICC protocol. Imaging was conducted with a Leica SPE confocal laser scanning microscope where cells were repeatedly imaged with the 635nm laser at 100% power over 84 seconds of continuous exposure to induce photobleaching. Average intensity values were calculated from images then data was normalised to the first exposure frame to generate normalised intensity. Statistics: decay curve: F-test, F_1,896=1315, p<0.0001, final intensity: two tailed t-test, t₈=10.74, p<0.0001, initial brightness: two tailed t-test, t₈=0.13, p=0.90

Rat cerebellum stained for myelin basic protein (MBP, HB8014) and calretinin (HB6494) using HB7393 goat anti-mouse IgG H&L (Janelia Fluor® 646) preadsorbed and HB9317 goat anti-rabbit IgG H&L (AF488) secondary antibodies. 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 HB8014 (1:2,000 dilution, 0.5µg/ml) and HB6494 (1:2,000 dilution). This was followed by a two-hour incubation with HB7393 goat anti-mouse IgG H&L (Janelia Fluor® 646) preadsorbed and HB9317 goat anti-rabbit IgG H&L (AF488) secondary antibodies at a 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 DMI6000B fluorescence microscope with a 20x objective using DAP (40ms exposure), L5 (200ms exposure) and Y5 (220ms exposure) filters. Following capture, deconvolution was carried out using Huygens Professional before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Rat cerebellum stained for β3-tubulin (HB6639) using HB7393 goat anti-mouse IgG H&L (Janelia Fluor® 646) preadsorbed secondary antibody. 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 HB6639 (1:1,000 dilution, 1µg/ml). This was followed by a two-hour incubation with HB7393 goat anti-mouse IgG H&L (Janelia Fluor® 646) preadsorbed secondary antibody at a 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 as a z-stack (0.3µm spacing) using a Leica DMI6000B fluorescence microscope with a 40x objective using DAP (5ms exposure) and Y5 (100ms exposure) filters. Following capture, deconvolution was carried out using Huygens Professional before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Rat cerebellum stained for myelin basic protein (MBP, HB8014) using HB7393 goat anti-mouse IgG H&L (Janelia Fluor® 646) preadsorbed secondary antibody. 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 HB8014 (1:2,000 dilution, 0.5µg/ml). This was followed by a two-hour incubation with HB7393 goat anti-mouse IgG H&L (Janelia Fluor® 646) preadsorbed secondary antibody at a 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 DMI6000B fluorescence microscope with a 20x objective using DAP (40ms exposure) and Y5 (220ms exposure) filters. Following capture, deconvolution was carried out using Huygens Professional before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Rat cerebellum stained for myelin basic protein (MBP, HB8014) and calretinin (HB6494) using HB7393 goat anti-mouse IgG H&L (Janelia Fluor® 646) preadsorbed and HB9317 goat anti-rabbit IgG H&L (AF488) secondary antibodies. 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 HB8014 (1:2,000 dilution, 0.5µg/ml) and HB6494 (1:2,000 dilution). This was followed by a two-hour incubation with HB7393 goat anti-mouse IgG H&L (Janelia Fluor® 646) preadsorbed and HB9317 goat anti-rabbit IgG H&L (AF488) secondary antibodies at a 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 DMI6000B fluorescence microscope with a 20x objective using DAP (40ms exposure), L5 (200ms exposure) and Y5 (220ms exposure) filters. Following capture, deconvolution was carried out using Huygens Professional before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).