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

Rat midbrain stained for Neurofilament L (NfL, HB7266) and parvalbumin (PV, HB6457) using HB8283 goat anti-rabbit IgG H&L (Janelia Fluor® 646) preadsorbed and HB8529 goat anti-mouse IgG H&L (AF488) preadsorbed 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 HB7266 (1:4,000 dilution, 0.25µg/ml) and HB6457 (1:4,000 dilution, 0.25µg/ml). This was followed by a two-hour incubation with HB8283 goat anti-rabbit IgG H&L (Janelia Fluor® 646) preadsorbed and HB8529 goat anti-mouse IgG H&L (AF488) preadsorbed 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 10x objective using DAP (80ms exposure), L5 (270ms exposure) and Y5 (350ms 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 hippocampus CA1 stained for Neurofilament L (NfL, HB7266) and parvalbumin (PV, HB6457) using HB8283 goat anti-rabbit IgG H&L (Janelia Fluor® 646) preadsorbed and HB8529 goat anti-mouse IgG H&L (AF488) preadsorbed 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 HB7266 (1:4,000 dilution, 0.25µg/ml) and HB6457 (1:4,000 dilution, 0.25µg/ml). This was followed by a two-hour incubation with HB8283 goat anti-rabbit IgG H&L (Janelia Fluor® 646) preadsorbed and HB8529 goat anti-mouse IgG H&L (AF488) preadsorbed 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 40x objective using DAP (8ms exposure), L5 (40ms exposure) and Y5 (157ms 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 midbrain stained for Tyrosine hydroxylase (TH, HB7167) and GFAP (HB8001) using HB8283 goat anti-rabbit IgG H&L (Janelia Fluor® 646) preadsorbed and HB8529 goat anti-mouse IgG H&L (AF488) preadsorbed 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 HB8001 (1:4,000 dilution) and HB7167 (1:4,000 dilution). This was followed by a two-hour incubation with HB8283 goat anti-rabbit IgG H&L (Janelia Fluor® 646) preadsorbed and HB8529 goat anti-mouse IgG H&L (AF488) preadsorbed 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. Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured in Lightning deconvolution mode using a 40x objective (2.25x zoom), 405nm (15.5% power, PMT: 723.5V gain), 488nm (1.5% power, Hyd: 10% gain) and 633nm (0.5% power, Hyd: 10% gain) laser lines in a z-stack (0.54µm spacing). The stack was 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

HEK293T cells stained with for Mitochondrial Fission Factor (MFF) using HB8283 goat anti-rabbit 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. An anti-MFF antibody was incubated overnight (4°C) at a 1:1,000 dilution followed by a one hour incubation with HB8283 goat anti-rabbit 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 using a Leica DMI6000B inverted epifluorescence microscope with a 40x objective and DAP (6ms exposure) / Y5 (450ms exposure) filters. Images were captured as a z-stack (0.3µm spacing) before being deconvolved using Huygens Essential (Scientific Volume Imagine) and flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Rat midbrain stained for Tyrosine hydroxylase (TH, HB7167) and GFAP (HB8001) using HB8283 goat anti-rabbit IgG H&L (Janelia Fluor® 646) preadsorbed and HB8529 goat anti-mouse IgG H&L (AF488) preadsorbed 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 HB8001 (1:4,000 dilution) and HB7167 (1:4,000 dilution). This was followed by a two-hour incubation with HB8283 goat anti-rabbit IgG H&L (Janelia Fluor® 646) preadsorbed and HB8529 goat anti-mouse IgG H&L (AF488) preadsorbed 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 (20ms exposure), L5 (61ms exposure) and RHO (250ms 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 hippocampus stained for Neurofilament L (NfL, HB7266) and parvalbumin (PV, HB6457) using HB8283 goat anti-rabbit IgG H&L (Janelia Fluor® 646) preadsorbed and HB8529 goat anti-mouse IgG H&L (AF488) preadsorbed 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 HB7266 (1:4,000 dilution, 0.25µg/ml) and HB6457 (1:4,000 dilution, 0.25µg/ml). This was followed by a two-hour incubation with HB8283 goat anti-rabbit IgG H&L (Janelia Fluor® 646) preadsorbed and HB8529 goat anti-mouse IgG H&L (AF488) preadsorbed 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 (45ms exposure), L5 (270ms exposure) and Y5 (300ms 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 corpus callosum stained for Neurofilament L (NfL, HB7266) using HB8283 goat anti-rabbit IgG H&L (Janelia Fluor® 646) preadsorbed as the 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 HB7266 (1:4,000 dilution, 0.25µg/ml). This was followed by a two-hour incubation with HB8283 goat anti-rabbit 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 10x objective using DAP (80ms exposure) and Y5 (350ms 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 midbrain stained for Tyrosine hydroxylase (TH, HB7167) and GFAP (HB8001) using HB8283 goat anti-rabbit IgG H&L (Janelia Fluor® 646) preadsorbed and HB8529 goat anti-mouse IgG H&L (AF488) preadsorbed 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 HB8001 (1:4,000 dilution) and HB7167 (1:4,000 dilution). This was followed by a two-hour incubation with HB8283 goat anti-rabbit IgG H&L (Janelia Fluor® 646) preadsorbed and HB8529 goat anti-mouse IgG H&L (AF488) preadsorbed 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 40x objective using DAP (5ms exposure), L5 (27ms exposure) and Y5 (30ms 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).