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

Rat brain stained for GFAP (HB8267) and Neurofilament L (HB7266) using HB8144 goat anti-mouse IgG H&L (Janelia Fluor® 525) preadsorbed and goat anti-rabbit 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 HB8267 (1:1000, 1µg/ml) and HB7266 (1:4000 dilution, 0.25µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (HB8144 Goat Anti-Mouse IgG H&L (Janelia Fluor® 525) preadsorbed and goat anti-rabbit DyLight 650). 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 SP8 confocal microscope in Lightning mode with a 40x objective using 405nm (21.1% power, PMT: 592V gain), 514nm (1.0% power, Hyd: 10% gain) and 561nm (0.5% power, 22.7% gain) lasers.Following capture the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Rat brain stained for GFAP (HB8267) and Neurofilament L (HB7266) using HB8144 goat anti-mouse IgG H&L (Janelia Fluor® 525) preadsorbed and goat anti-rabbit 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 HB8267 (1:1000, 1µg/ml) and HB7266 (1:4000 dilution, 0.25µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (HB8144 Goat Anti-Mouse IgG H&L (Janelia Fluor® 525) preadsorbed and goat anti-rabbit DyLight 650). 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 SP8 confocal microscope in Lightning mode with a 40x objective using 405nm (21.1% power, PMT: 592V gain), 514nm (1.0% power, Hyd: 10% gain) and 561nm (0.5% power, 22.7% gain) lasers.Following capture the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Rat brain stained for GFAP (HB8267) and Neurofilament L (HB7266) using HB8144 goat anti-mouse IgG H&L (Janelia Fluor® 525) preadsorbed and goat anti-rabbit 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 HB8267 (1:1000, 1µg/ml) and HB7266 (1:4000 dilution, 0.25µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (HB8144 Goat Anti-Mouse IgG H&L (Janelia Fluor® 525) preadsorbed and goat anti-rabbit DyLight 650). 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 SP8 confocal microscope in Lightning mode with a 63x objective using 405nm (21.1% power, PMT: 670V gain), 514nm (2.2% power, Hyd: 17% gain) and 561nm (2.0% power, 23% gain) lasers.Following capture 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 HB6497 for Vimentin 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. HB6497 was incubated overnight (4°C) at a 1:2,000 dilution (0.5µg/ml) followed by a one hour incubation with HB8144 goat anti-mouse 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 (1ms exposure) / RHO (10ms 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).