Goat Anti-Mouse IgG H&L (Biotin) preadsorbed ValidAbTM

Rat hippocampus stained with HB6457 to detect Parvalbumin and HB6494 to detect Calretinin. Parvalbumin was detected using an anti-mouse biotin antibody followed by incubation with Streptavidin Janelia Fluor® 525 HB15382.

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% donkey serum before incubation overnight in Parvalbumin HB6457 (1:1000 dilution) and Calretinin HB6494 (1:4000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-mouse biotin antibody (HB11345), at a dilution of 1:250 and goat anti-rabbit Dylight™ 650 (Thermofisher) at a dilution of 1:300. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 525 HB15382 at 1.0 µg/mL for 2 hours at room temperature. 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. Images were captured as a tilescan using a 20x objective in a z-stack with exposures: A4: 57.803 ms, Y3: 599.044 ms, Y5 599.044 ms. Stacks were deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Rat hippocampus stained with HB6457 to detect Parvalbumin and HB6406 to detect GFAP. Parvalbumin was detected using an anti-mouse biotin antibody followed by incubation with Streptavidin Janelia Fluor® 525 HB12382.

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% donkey serum before incubation overnight in Parvalbumin HB6457 (1:1000 dilution) and GFAP HB6406 (1:4000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-mouse biotin antibody at a dilution of 1:250 and goat anti-chicken Alexa Fluor™ 488 (Invitrogen) at a dilution of 1:300. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 525 HB12382 at 1.0 µg/mL for 2 hours at room temperature. DAPI HB0747 was used at 1 µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured using a 20x objective in a z-stack with exposures: A4: 57.803 ms, GFP: 524.511 ms, Y3: 524.211 ms Stacks were deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Rat hippocampus stained with HB6457 to detect Parvalbumin and HB6406 to detect GFAP. Parvalbumin was detected using an anti-mouse biotin antibody HB11345 followed by incubation with Streptavidin Janelia Fluor® HB18064.

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% donkey serum before incubation overnight in Parvalbumin HB6457 (1:1,000 dilution) and GFAP HB6406 (1:4000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-mouse biotin antibody at a dilution of 1:250 and goat anti-chicken Alexa Fluor™ 488, (Invitrogen) at a dilution of 1:300. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 549 HB18064 at 1.0 µg/mL for 2 hours at room temperature. DAPI HB0747 IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured using a 20x objective in a z-stack with exposures: DAP: 224.256 ms, GFP: 677.506 ms, Y3: 1044.272 ms Stacks were deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Rat hippocampus stained with HB6457 to detect Parvalbumin and HB6406 to detect GFAP. Parvalbumin was detected using anti-mouse biotin antibody HB11345 followed by incubation with Streptavidin AF488 HB13531. 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% donkey serum before incubation overnight in Parvalbumin HB6457 (1:1000 dilution) and GFAP HB6406 (1:4000) at 4°C. This was followed by a two hour incubation with goat anti-mouse biotin antibody HB11345 at a dilution of 1:250 and goat anti-chicken Alex Fluor™ 647 (Invitrogen) at a dilution of 1:300. Following three washes in PBST, sections were incubated with Streptavidin AF488 HB13531 at 1.0 µL/mL for 2 hours at room temperature. 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. Images were captured using a 20x objective in a z-stack with exposures: A4: 57.803 ms, GFP: 151.803 ms, Y5: 599.044 ms Stacks were deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).