Goat Anti-Rabbit IgG H&L (Janelia Fluor® 525) preadsorbed ValidAb™

Rat striatum stained for Tyrosine hydroxylase (TH, HB6605) and NeuN (HB6429) using HB6813 goat anti-rabbit IgG H&L (Janelia Fluor® 525) preadsorbed and goat anti-mouse DyLight 488 conjugated 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 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6605 (1:1,000 dilution) and HB6429 (1:1,000 dilution, 1µg/ml). This was followed by a two hour incubation with HB6813 Goat Anti-Rabbit IgG H&L (Janelia Fluor® 525) preadsorbed and goat anti-mouse DyLight 488 conjugated 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 (8ms exposure), L5 (200ms 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 striatum stained for Tyrosine hydroxylase (TH, HB6605) and NeuN (HB6429) using HB6813 goat anti-rabbit IgG H&L (Janelia Fluor® 525) preadsorbed and goat anti-mouse DyLight 488 conjugated 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 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6605 (1:1,000 dilution) and HB6429 (1:1,000 dilution, 1µg/ml). This was followed by a two hour incubation with HB6813 Goat Anti-Rabbit IgG H&L (Janelia Fluor® 525) preadsorbed and goat anti-mouse DyLight 488 conjugated 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 (8ms exposure), L5 (100ms exposure) and RHO (125ms 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 striatum stained for Tyrosine hydroxylase (TH, HB6605) and NeuN (HB6429) using HB6813 goat anti-rabbit IgG H&L (Janelia Fluor® 525) preadsorbed and goat anti-mouse DyLight 488 conjugated 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 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6605 (1:1,000 dilution) and HB6429 (1:1,000 dilution, 1µg/ml). This was followed by a two hour incubation with HB6813 Goat Anti-Rabbit IgG H&L (Janelia Fluor® 525) preadsorbed and goat anti-mouse DyLight 488 conjugated 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 (13ms exposure), L5 (200ms 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).

HEK293T cells stained with for Lamin B1 using HB8144 goat anti-mouse IgG H&L (Janelia Fluor® 525) preadsorbed 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-Lamin B1 antibody was incubated overnight (4°C) at a 1:2,000 dilution followed by a one hour incubation with HB6813 Goat Anti-Rabbit IgG H&L (Janelia Fluor® 525) preadsorbed secondary antibody (1:3,00 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 100x objective and DAP (2ms exposure) / RHO (50ms exposure) filters. Images were captured as a z-stack (0.22µm 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).