Anti-Calbindin antibody ValidAb™
(HB6396)
Product overview
| Name | Anti-Calbindin antibody ValidAb™ |
| Host | Mouse |
| Clonality | Monoclonal |
| Target | Calbindin |
| Description | Antibody to Calbindin - calcium binding protein used as a marker for an inhibitory interneuron subtype. Part of the ValidAb™ range of highly validated, data-rich antibodies. |
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Validation data
Figure 1. Calbindin and Calretinin expression in rat cerebellum
HB6396 (mouse monoclonal anti-calbindin) and HB6494 (rabbit polyclonal anti-calretinin) stain differing populations of cells in rat cerebellum. 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 HB6396 (1:1,000 dilution, 1µg/ml) and HB6494 (1:4,000 dilution). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse DyLight 488, Thermofisher 35503 and polyclonal goat anti-rabbit DyLight 594, Thermofisher 35561). 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 connected to a Photometric Prime 95B camera. The image was captured using a 40x objective (DAPI: 10ms, L5: 7.5ms, TX2: 19.7ms exposures) in a z-stack (1.0µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Calbindin and Calretinin expression in rat hippocampus CA1
HB6396 (mouse monoclonal anti-calbindin) and HB6494 (rabbit polyclonal anti-calretinin) stain differing populations of cells in rat cerebellum. 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 HB6396 (1:1,000 dilution, 1µg/ml) and HB6494 (1:4,000 dilution). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse DyLight 488, Thermofisher 35503 and polyclonal goat anti-rabbit DyLight 594, Thermofisher 35561). 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 connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 40x objective (DAPI: 26ms, L5: 133ms, TX2: 28ms exposures) in a z-stack (1.0µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. Concentration response of HB6396 and HB6494 in rat cerebellum.
HB6396 (mouse monoclonal anti-calbindin) and HB6494 (rabbit polyclonal anti-calretinin) show successful staining in the cerebellum at dilutions as low as 1:4,000 (calbindin) and 1:16,000 (calretinin). 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 HB6396 (1:500 to 1:4,000 dilutions, 0.25 - 2µg/ml) and HB6494 (1:2,000 to 1:16,000 dilutions). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse DyLight 488, Thermofisher 35503 and polyclonal goat anti-rabbit DyLight 594, Thermofisher 35561). 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 connected to a Photometric Prime 95B camera. Images were captured using a 20x objective in a z-stack. Images were deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682). Exposure times were as follows:
- Calbindin (1:500), Calretinin (1:2,000): DAPI: 80ms, GFP: 211ms, Y3: 396ms.
- Calbindin (1:1,000), Calretinin (1:4,000): DAPI: 80ms, GFP: 211ms, Y3: 396ms.
- Calbindin (1:2,000), Calretinin (1:8,000): DAPI: 80ms, GFP: 445ms, Y3: 396ms.
- Calbindin (1:4,000), Calretinin (1:16,000): DAPI: 80ms, GFP: 286ms, Y3: 639ms.
Figure 4. VGAT, Calbindin and GFAP expression in rat cerebellum.
Rat cerebellum stained by HB6406 for GFAP (chicken polyclonal), HB6396 for Calbindin (mouse monoclonal) and HB6714 for VGAT (rabbit polyclonal). 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 HB6406 (1:2000 dilution), HB6396 (1:2000 dilution) and HB6714 (1:1000 dilution, 0.25µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948; polyclonal goat anti-mouse Janelia Fluor 549; Goat anti-rabbit DyLight 650, Thermofisher 11804574). 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 connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 20x objective (DAPI: 25ms, GFP: 163.7ms, Y3: 373.1ms, Y5: 1.0s exposures) in a z-stack (2.4µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 5. VGAT, Calbindin and GFAP expression in rat cerebellum.
Rat cerebellum stained by HB6406 for GFAP (chicken polyclonal), HB6396 for Calbindin (mouse monoclonal) and HB6714 for VGAT (rabbit polyclonal). 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 HB6406 (1:2000 dilution), HB6396 (1:2000 dilution) and HB6714 (1:1000 dilution, 0.25µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948; polyclonal goat anti-mouse Janelia Fluor 549; Goat anti-rabbit DyLight 650, Thermofisher 11804574). 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 connected to a Photometric Prime 95B camera. The image was captured using a 100x objective (DAPI: 7.5ms, GFP: 63.9ms, Y3: 43.1ms, Y5: 185.9s exposures) in a z-stack (0.2µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 6. Calbindin and Calretinin positive neurons in rat cerebellum
HB6396 (mouse monoclonal anti-calbindin) and HB6494 (rabbit polyclonal anti-calretinin) stain differing populations of cells in rat cerebellum. 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 HB6396 (1:500 dilution, 2µg/ml) and HB6494 (1:2,000 dilution). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse DyLight 488, Thermofisher 35503 and polyclonal goat anti-rabbit DyLight 594, Thermofisher 35561). 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 connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 200x objective (DAPI: 80ms, GFP: 211ms, Y3: 396ms exposures) in a z-stack (2.4µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 1. Calbindin and Calretinin expression in rat cerebellum
HB6396 (mouse monoclonal anti-calbindin) and HB6494 (rabbit polyclonal anti-calretinin) stain differing populations of cells in rat cerebellum. 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 HB6396 (1:1,000 dilution, 1µg/ml) and HB6494 (1:4,000 dilution). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse DyLight 488, Thermofisher 35503 and polyclonal goat anti-rabbit DyLight 594, Thermofisher 35561). 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 connected to a Photometric Prime 95B camera. The image was captured using a 40x objective (DAPI: 10ms, L5: 7.5ms, TX2: 19.7ms exposures) in a z-stack (1.0µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Calbindin and Calretinin expression in rat hippocampus CA1
HB6396 (mouse monoclonal anti-calbindin) and HB6494 (rabbit polyclonal anti-calretinin) stain differing populations of cells in rat cerebellum. 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 HB6396 (1:1,000 dilution, 1µg/ml) and HB6494 (1:4,000 dilution). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse DyLight 488, Thermofisher 35503 and polyclonal goat anti-rabbit DyLight 594, Thermofisher 35561). 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 connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 40x objective (DAPI: 26ms, L5: 133ms, TX2: 28ms exposures) in a z-stack (1.0µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. Concentration response of HB6396 and HB6494 in rat cerebellum.
HB6396 (mouse monoclonal anti-calbindin) and HB6494 (rabbit polyclonal anti-calretinin) show successful staining in the cerebellum at dilutions as low as 1:4,000 (calbindin) and 1:16,000 (calretinin). 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 HB6396 (1:500 to 1:4,000 dilutions, 0.25 - 2µg/ml) and HB6494 (1:2,000 to 1:16,000 dilutions). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse DyLight 488, Thermofisher 35503 and polyclonal goat anti-rabbit DyLight 594, Thermofisher 35561). 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 connected to a Photometric Prime 95B camera. Images were captured using a 20x objective in a z-stack. Images were deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682). Exposure times were as follows:
- Calbindin (1:500), Calretinin (1:2,000): DAPI: 80ms, GFP: 211ms, Y3: 396ms.
- Calbindin (1:1,000), Calretinin (1:4,000): DAPI: 80ms, GFP: 211ms, Y3: 396ms.
- Calbindin (1:2,000), Calretinin (1:8,000): DAPI: 80ms, GFP: 445ms, Y3: 396ms.
- Calbindin (1:4,000), Calretinin (1:16,000): DAPI: 80ms, GFP: 286ms, Y3: 639ms.
Figure 4. VGAT, Calbindin and GFAP expression in rat cerebellum.
Rat cerebellum stained by HB6406 for GFAP (chicken polyclonal), HB6396 for Calbindin (mouse monoclonal) and HB6714 for VGAT (rabbit polyclonal). 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 HB6406 (1:2000 dilution), HB6396 (1:2000 dilution) and HB6714 (1:1000 dilution, 0.25µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948; polyclonal goat anti-mouse Janelia Fluor 549; Goat anti-rabbit DyLight 650, Thermofisher 11804574). 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 connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 20x objective (DAPI: 25ms, GFP: 163.7ms, Y3: 373.1ms, Y5: 1.0s exposures) in a z-stack (2.4µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 5. VGAT, Calbindin and GFAP expression in rat cerebellum.
Rat cerebellum stained by HB6406 for GFAP (chicken polyclonal), HB6396 for Calbindin (mouse monoclonal) and HB6714 for VGAT (rabbit polyclonal). 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 HB6406 (1:2000 dilution), HB6396 (1:2000 dilution) and HB6714 (1:1000 dilution, 0.25µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948; polyclonal goat anti-mouse Janelia Fluor 549; Goat anti-rabbit DyLight 650, Thermofisher 11804574). 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 connected to a Photometric Prime 95B camera. The image was captured using a 100x objective (DAPI: 7.5ms, GFP: 63.9ms, Y3: 43.1ms, Y5: 185.9s exposures) in a z-stack (0.2µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 6. Calbindin and Calretinin positive neurons in rat cerebellum
HB6396 (mouse monoclonal anti-calbindin) and HB6494 (rabbit polyclonal anti-calretinin) stain differing populations of cells in rat cerebellum. 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 HB6396 (1:500 dilution, 2µg/ml) and HB6494 (1:2,000 dilution). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse DyLight 488, Thermofisher 35503 and polyclonal goat anti-rabbit DyLight 594, Thermofisher 35561). 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 connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 200x objective (DAPI: 80ms, GFP: 211ms, Y3: 396ms exposures) in a z-stack (2.4µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Product information
| Immunogen | Recombinant human calbindin expressed in and purified from E. coli. |
| Clone number | 5A9 |
| Isotype | IgG2a |
| Purification | Protein G affinity chromatography |
| Concentration | 1mg/ml |
| Formulation | 50% PBS, 50% glycerol + 5mM sodium azide |
| Predicted species reactivity | Mouse, Rat, Human, Cow |
| Tested species reactivity | Rat |
Tested applications
| Applications | IHC(IF) |
| IHC(IF) optimal concentration | 0.25µg/ml (1:4,000) as tested in free-floating paraformaldehyde fixed rat cerebellum sections |
| Positive control | Calbindin is strongly expressed in a subset of inhibitory interneurones in the brain alongside in distal tubules of the kidney. |
| Negative control | Calbindin expression is absent in most non-neural tissues such as in liver, muscle and lung. |
| Open data link | Please follow this link to OSF |
Target information
| Other names | CALB1, CALB, Calbindin 1, D-28K |
| UniProt ID | P05937 |
| Structure image |  |
| Gene name | CALB1 |
| NCBI full gene name | calbindin 1 |
| Entrez gene ID |
| Amino acids | 261 (30.0kDa) |
| Isoforms | Calbindin has two described isoforms:
|
| Expression | Calbindin is expressed in inhibitory interneurones in the brain with particularly high expression in the cerebellum and cortex alongside also being expressed in the kidney (collecting ducts and distal tubules) and retina. |
| Subcellular expression | Calbindin is primarily expressed in the cytosol of expressing cells with expression also having being reported in the nucleus. |
| Processing | Calbindin has the initiator methionine removed before forming a final conformation. |
| Post translational modifications | Calbindin is acetylated on alanine 2. |
| Homology (compared to human) | Mouse and rat calbindin have 98.5% identity with human calbindin. Mouse and rat calbindin show 99.2% homology (S60T and T232S). |
| Similar proteins | In a BLAST search Calretinin (58.5% identity, 29kDa) was the only protein identified with significant homology to Calbindin. |
Storage & Handling
| Storage instructions | -20°C |
| Important | This product is for RESEARCH USE ONLY and is not intended for therapeutic or diagnostic use. Not for human or veterinary use |
Technical guides
Western Blot Protocol (1 MB) Immunocytochemistry Protocol (1.2 MB) Immunohistochemistry Protocol (1.6 MB) References for Anti-Calbindin antibody ValidAb™
References are publications that support the biological activity of the product
-
Densities and numbers of calbindin and parvalbumin positive neurons across the rat and mouse brain.
Bjerke IE et al (2021) iScience 24 : 101906 -
Crucial role of calbindin-D28k in the pathogenesis of Alzheimer's disease mouse model.
Kook SY et al (2014) Cell death and differentiation 21 : 1575-87 -
Three functional facets of calbindin D-28k.
Schmidt H (2012) Frontiers in molecular neuroscience 5 : 25 -
Biological actions and mechanism of action of calbindin in the process of apoptosis.
Christakos S et al (2004) The Journal of steroid biochemistry and molecular biology 89-90 : 401-4 -
Calbindin in cerebellar Purkinje cells is a critical determinant of the precision of motor coordination.
Barski JJ et al (2003) The Journal of neuroscience : the official journal of the Society for Neuroscience 23 : 3469-77
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