Goat Anti-Rabbit IgG H&L (Biotin) preadsorbed ValidAb^TM

Rabbit polyclonal anti-calretinin HB6494 staining interneurons and chicken polyclonal anti-GFAP a href="/anti-gfap-antibody-chicken-validab.html">HB6406 staining of astrocytes in the rat hippocampus. Calretinin was detected using an anti-rabbit biotin antibody (HB11036) followed by incubation with Streptavidin Janelia Fluor® 646 HB17045.

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 anti-calretinin HB6494 (1:1,000 dilution) and anti-GFAP HB6406 (1:4000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-rabbit biotin antibody (HB11036) 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® 646 HB17045 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 SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured 10x objective, 405nm (6.0% power, PMT: 725.1V gain), 488nm (2.18% power, Hyd: 22.9% gain) and 633nm (10.16% power, Hyd: 94.7% gain) laser lines in a z-stack (3.67µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

HB11036 (goat anti-rabbit H&L biotin antibody) in combination with HB6605 (rabbit polyclonal anti-tyrosine hydroxylase antibody) and HB5255 (Streptavidin HRP) was used to stain the dense network of dopaminergic terminals in the rat caudate putamen. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 70% ethanol and embedded in paraffin. Deparaffinization was carried out following cutting of the tissue block into 5µm sections. Antigen retrieval was carried out using citrate buffer (HB8687) at 95°C for 20 minutes before sections were blocked in 1% BSA, 10% serum. HB6605 was incubated overnight (4°C) at a 1:500 dilution. Following washing and peroxide blocking with 0.3% H₂O₂, the sections were incubated in HB11036 (goat anti-rabbit H&L biotin antibody) for 1 hour at room temperature (1:300 dilution). Following subsequent washes, the sections were incubated in HB5255 (Streptavidin HRP) for 30 minutes at room temperature (1:500 dilution). The reaction was developed using DAB (HB0687) and counterstained using hematoxylin (HB6189) before sections were dehydrated and mounted using DPX. For more details please see our paraffin embedded immunohistochemistry protocol. Images were taken on a standard widefield microscope using brightfield illumination and a 5x objective.

Rabbit polyclonal anti-Calretinin HB6494 and mouse monoclonal anti-Parvalbumin HB6457 staining of different interneuron populations in the rat hippocampus. Calretinin was detected using an anti-rabbit biotin antibody (HB11036) followed by incubation with Streptavidin Janelia Fluor® 646 HB17045.

Method: Rat brains were dissected and fixed overnight in 4% PFA before incubation 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 anti-calretinin HB6494 (1:4,000 dilution) and anti-parvalbumin HB6457 (1:1000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-rabbit biotin antibody (HB11036) at a dilution of 1:250, and goat anti-mouse Dylight™ 594 (Thermofisher) at a dilution of 1:300. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 646 HB6494 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 SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured using a 10x objective, 405nm (6.0% power, PMT: 725.1V gain), 580nm (3.63% power, Hyd: 68% gain) and 633nm (10.16% power, Hyd: 94.7% gain) laser lines in a z-stack (3.67µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

HB11036 (goat anti-rabbit H&L biotin antibody) in combination with HB6605 (rabbit polyclonal anti-tyrosine hydroxylase antibody) and HB5255 (Streptavidin HRP) were used to stain dopaminergic terminals in the rat striatum. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 70% ethanol and embedded in paraffin. Deparaffinization was carried out following cutting of the tissue block into 5µm sections. Antigen retrieval was carried out using citrate buffer (HB8687) at 95°C for 20 minutes before sections were blocked in 1% BSA, 10% serum. HB6605 was incubated overnight (4°C) at a 1:500 dilution. Following washing and peroxide blocking with 0.3% H₂O₂, the sections were incubated in HB11036 (goat anti-rabbit H&L biotin antibody) for 1 hour at room temperature (1:300 dilution). Following subsequent washes, the sections were incubated in HB5255 (Streptavidin HRP) for 30 minutes at room temperature (1:2,000 dilution). The reaction was developed using DAB (HB0687) and counterstained using hematoxylin (HB6189) before sections were dehydrated and mounted using DPX. For more details please see our paraffin embedded immunohistochemistry protocol. Images were taken on a standard widefield microscope using brightfield illumination and a 5x objective.

HB17045 produces optimal staining of Calretinin positive interneurons in the hippocampus at 1 µg/mL.

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 anti-parvalbumin HB6494 (1:4,000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-rabbit biotin antibody HB11345 at a dilution of 1:250. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 646 HB17045) at 0.5 µg/mL, 1.0 µg/mL or 5.0 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 using a 20x objective in a z-stack with exposures:

• 0.5 µg/mL: DAP: 46.156 ms, Y5: 11222.92 ms
• 1.0 µg/mL: DAP: 46.156 ms, Y5: 6131.079 ms
• 5.0 µg/mL: DAP: .756 ms, Y5: 11222.92 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).

HB11036 (goat anti-rabbit H&L biotin antibody) in combination with HB6605 (rabbit polyclonal anti-tyrosine hydroxylase antibody) and HB5255 (Streptavidin HRP) were used to stain dopaminergic terminals in the rat caudate putamen. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 70% ethanol and embedded in paraffin. Deparaffinization was carried out following cutting of the tissue block into 5µm sections. Antigen retrieval was carried out using citrate buffer (HB8687) at 95°C for 20 minutes before sections were blocked in 1% BSA, 10% serum. HB6605 was incubated overnight (4°C) at a 1:500 dilution. Following washing and peroxide blocking with 0.3% H₂O₂, the sections were incubated in HB11036 (goat anti-rabbit H&L biotin antibody) for 1 hour at room temperature (1:300 dilution). Following subsequent washes the sections were incubated in HB5255 (Streptavidin HRP) for 30 minutes at room temperature (1:1,000 dilution). The reaction was developed using DAB (HB0687) and counterstained using hematoxylin (HB6189) before sections were dehydrated and mounted using DPX. For more details please see our paraffin embedded immunohistochemistry protocol. Images were taken on a standard widefield microscope using brightfield illumination and a 20x objective.