Anti-Calretinin antibody ValidAbTM
(HB6494)
Product overview
| Name | Anti-Calretinin antibody ValidAbTM |
| Host | Rabbit |
| Clonality | Polyclonal |
| Target | Calretinin |
| Description | Antibody to Calretinin - 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. Calretinin and Parvalbumin expressing interneurons in the rat hippocampus
Rat hippocampus stained by HB6457 for Parvalbumin (mouse monoclonal) and HB6494 for Calretinin (rabbit polyclonal) showing the different populations of interneurons in the hippocampus. 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 HB6457 (1:1000, 1µg/ml) and HB6494 (1:4000 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 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. The image was captured as a tilescan using a 10x objective in a z-stack (6.1µm spacing). The image was captured using DAP (205.0ms exposure), L5 (444.3ms exposure) and TX2 (231.4ms exposure) filters. The stack was deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Calretinin and Calbindin 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. Calretinin and Parvalbumin expressing interneurons in hippocampal CA1.
Rat hippocampal CA1 stained by HB6457 for Parvalbumin (mouse monoclonal) and HB6494 for Calretinin (rabbit polyclonal) showing the different populations of interneurons in the hippocampus. 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 HB6457 (1:1000, 1µg/ml) and HB6494 (1:4000 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 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. The image was captured as a tilescan using a 40x objective in a z-stack (1.0µm spacing). The image was captured using DAP (95.0ms exposure), L5 (363ms exposure) and TX2 (136.1ms exposure) filters. The stack was deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 4. Parvalbumin and Calretinin expressing interneurons in hippocampal CA1.
A section of hippocampal CA1 stained by HB6457 for Parvalbumin (mouse monoclonal) and HB6494 for Calretinin (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 HB6457 (1:1000, 1µg/ml) and HB6494 (1:4000 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 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 SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured in as a tilescan in Lightning deconvolution mode using a 63x objective (2.25x zoom), 405nm (22.3% power, PMT: 681V gain), 488nm (2.2% power, Hyd: 10% gain) and 561nm (2.1% power, Hyd: 20.8% gain) laser lines in a z-stack (0.78µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 5. 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 6. 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 7. Calretinin expression in various tissue lysates and preparations.
HB494 revealed the ≈26kDa band associated with Calretinin only in neural tissue samples. Method: mouse brain and rat brain membrane (P2) and cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Other tissue lysates were prepared following established protocols from freshly dissected tissue (see our guide on WB sample preparation). Samples were loaded (20µg / lane) onto a 20% acrylamide gel alongside a protein ladder (NEB Prestained broad range) before being run at 60V for 30 minutes followed by 130V for 90 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6494 at a 1:4,000 dilution. Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-rabbit HRP conjugated, Sigma, A6154) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 6 min exposure, 700nm channel: 30 sec exposure). Following imaging the membrane was stripped with two changes of stripping buffer (HB7756) before being washed, blocked for 2 hours in 5% non-fat dry milk and incubated in HB9177 (mouse monoclonal anti-GAPDH, 1:4,000 dilution, 0.25µg/ml) overnight at 4°C. Following washing the membrane was incubated in a 1:10,000 dilution of a polyclonal goat anti-mouse HRP conjugated secondary antibody (Sigma Aldrich A3682) for 2hrs and visualised again using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 4 min exposure, 700nm channel: 30 sec exposure).
Figure 8. Concentration response of HB6494 staining in a rat brain cytosol preparation.
HB6494 produces reliable results down to a 1:64,000 dilution. Method: Cytosol fractions were prepared from fresh rat brains following established protocols (Molnar et al., 1993. Neuroscience 53:307-326). Samples were loaded (20µg / lane) onto a 20% acrylamide gel alongside a protein ladder (NEB Prestained broad range) before being run at 60V for 30 minutes followed by 130V for 90 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. Following transfer the membrane was cut into strips using Ponceau dye to visualise and cut individual lanes. Strips were blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6494. Each strip was incubated separately with a separate HB6494 concentration with this ranging from 1:1,000 to 1:256,000 dilutions. Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-rabbit HRP conjugated, Sigma, A6154) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure). Band intensity was calculated using Image Studio version 5.2.5 (LiCor) and a graph was constructed in GraphPad Prism 9 using a 3-parameter Hill equation curve fit.
Figure 9. Calretinin and Parvalbumin expressing neurons in rat cerebellum.
Rat cerebellum stained by HB6457 for Parvalbumin (mouse monoclonal) and HB6494 for Calretinin (rabbit polyclonal) showing the different populations of interneurons in the 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% donkey serum before incubation overnight in HB6457 (1:1000, 1µg/ml) and HB6494 (1:4000 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 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. The image was captured as a tilescan using a 40x objective in a z-stack (1.0µm spacing). The image was captured using DAP (95.0ms exposure), L5 (363.0ms exposure) and TX2 (201.3ms exposure) filters. The stack was deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 10. Calretinin and Calbindin 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 11. Calretinin and Parvalbumin expressing interneurons in hippocampal CA1.
A section of hippocampal CA1 stained by HB6457 for Parvalbumin (mouse monoclonal) and HB6494 for Calretinin (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 HB6457 (1:1000, 1µg/ml) and HB6494 (1:4000 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 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 SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured in Lightning deconvolution mode using a 20x objective (1.28x zoom), 405nm (22.3% power, PMT: 800V gain), 488nm (2.2% power, Hyd: 15.9% gain) and 561nm (2.1% power, Hyd: 20.8% gain) laser lines in a z-stack (0.86µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 1. Calretinin and Parvalbumin expressing interneurons in the rat hippocampus
Rat hippocampus stained by HB6457 for Parvalbumin (mouse monoclonal) and HB6494 for Calretinin (rabbit polyclonal) showing the different populations of interneurons in the hippocampus. 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 HB6457 (1:1000, 1µg/ml) and HB6494 (1:4000 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 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. The image was captured as a tilescan using a 10x objective in a z-stack (6.1µm spacing). The image was captured using DAP (205.0ms exposure), L5 (444.3ms exposure) and TX2 (231.4ms exposure) filters. The stack was deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Calretinin and Calbindin 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. Calretinin and Parvalbumin expressing interneurons in hippocampal CA1.
Rat hippocampal CA1 stained by HB6457 for Parvalbumin (mouse monoclonal) and HB6494 for Calretinin (rabbit polyclonal) showing the different populations of interneurons in the hippocampus. 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 HB6457 (1:1000, 1µg/ml) and HB6494 (1:4000 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 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. The image was captured as a tilescan using a 40x objective in a z-stack (1.0µm spacing). The image was captured using DAP (95.0ms exposure), L5 (363ms exposure) and TX2 (136.1ms exposure) filters. The stack was deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 4. Parvalbumin and Calretinin expressing interneurons in hippocampal CA1.
A section of hippocampal CA1 stained by HB6457 for Parvalbumin (mouse monoclonal) and HB6494 for Calretinin (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 HB6457 (1:1000, 1µg/ml) and HB6494 (1:4000 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 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 SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured in as a tilescan in Lightning deconvolution mode using a 63x objective (2.25x zoom), 405nm (22.3% power, PMT: 681V gain), 488nm (2.2% power, Hyd: 10% gain) and 561nm (2.1% power, Hyd: 20.8% gain) laser lines in a z-stack (0.78µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 5. 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 6. 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 7. Calretinin expression in various tissue lysates and preparations.
HB494 revealed the ≈26kDa band associated with Calretinin only in neural tissue samples. Method: mouse brain and rat brain membrane (P2) and cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Other tissue lysates were prepared following established protocols from freshly dissected tissue (see our guide on WB sample preparation). Samples were loaded (20µg / lane) onto a 20% acrylamide gel alongside a protein ladder (NEB Prestained broad range) before being run at 60V for 30 minutes followed by 130V for 90 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6494 at a 1:4,000 dilution. Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-rabbit HRP conjugated, Sigma, A6154) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 6 min exposure, 700nm channel: 30 sec exposure). Following imaging the membrane was stripped with two changes of stripping buffer (HB7756) before being washed, blocked for 2 hours in 5% non-fat dry milk and incubated in HB9177 (mouse monoclonal anti-GAPDH, 1:4,000 dilution, 0.25µg/ml) overnight at 4°C. Following washing the membrane was incubated in a 1:10,000 dilution of a polyclonal goat anti-mouse HRP conjugated secondary antibody (Sigma Aldrich A3682) for 2hrs and visualised again using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 4 min exposure, 700nm channel: 30 sec exposure).
Figure 8. Concentration response of HB6494 staining in a rat brain cytosol preparation.
HB6494 produces reliable results down to a 1:64,000 dilution. Method: Cytosol fractions were prepared from fresh rat brains following established protocols (Molnar et al., 1993. Neuroscience 53:307-326). Samples were loaded (20µg / lane) onto a 20% acrylamide gel alongside a protein ladder (NEB Prestained broad range) before being run at 60V for 30 minutes followed by 130V for 90 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. Following transfer the membrane was cut into strips using Ponceau dye to visualise and cut individual lanes. Strips were blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6494. Each strip was incubated separately with a separate HB6494 concentration with this ranging from 1:1,000 to 1:256,000 dilutions. Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-rabbit HRP conjugated, Sigma, A6154) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure). Band intensity was calculated using Image Studio version 5.2.5 (LiCor) and a graph was constructed in GraphPad Prism 9 using a 3-parameter Hill equation curve fit.
Figure 9. Calretinin and Parvalbumin expressing neurons in rat cerebellum.
Rat cerebellum stained by HB6457 for Parvalbumin (mouse monoclonal) and HB6494 for Calretinin (rabbit polyclonal) showing the different populations of interneurons in the 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% donkey serum before incubation overnight in HB6457 (1:1000, 1µg/ml) and HB6494 (1:4000 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 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. The image was captured as a tilescan using a 40x objective in a z-stack (1.0µm spacing). The image was captured using DAP (95.0ms exposure), L5 (363.0ms exposure) and TX2 (201.3ms exposure) filters. The stack was deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 10. Calretinin and Calbindin 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 11. Calretinin and Parvalbumin expressing interneurons in hippocampal CA1.
A section of hippocampal CA1 stained by HB6457 for Parvalbumin (mouse monoclonal) and HB6494 for Calretinin (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 HB6457 (1:1000, 1µg/ml) and HB6494 (1:4000 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 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 SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured in Lightning deconvolution mode using a 20x objective (1.28x zoom), 405nm (22.3% power, PMT: 800V gain), 488nm (2.2% power, Hyd: 15.9% gain) and 561nm (2.1% power, Hyd: 20.8% gain) laser lines in a z-stack (0.86µm spacing). 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 calretinin expressed in and purified from E. coli |
| Purification | Unpurified |
| Formulation | Lyophilised. When reconstituted contains serum with 1% recombinant BSA and 5mM sodium azide |
| Predicted species reactivity | Mouse, Rat, Human |
| Tested species reactivity | Mouse, Rat |
Tested applications
| Applications | WB, IHC(IF) |
| Western blot optimal concentration | A dilution of 1:8,000 as tested in a rat brain cytosol preparation. |
| IHC(IF) optimal concentration | A dilution of 1:4,000 as tested in paraformaldehyde fixed free-floating rat cerebellum sections. |
| Positive control | Calretinin is expressed in inhibitory interneurones in a wide range of brain regions including the cerebellum and hippocampus. Calretinin is also expressed in a wide array of cell lines (see the human protein atlas). |
| Negative control | Calretinin lacks expression in somatic tissues such as the liver, skin and skeletal muscle. Calretinin expression is also lacking in many cell lines such as HEK293T and HeLa. |
| Open data link | Please follow this link to OSF. |
Target information
| Other names | CALB2, CAB29, CAL2, Calbindin 2, 29kDa calbindin |
| UniProt ID | P22676 |
| Structure image |  |
| Gene name | CALB2 |
| NCBI full gene name | calbindin 2 |
| Entrez gene ID |
| Amino acids | 271 (31.5kDa) |
| Isoforms | Calretinin only has one described isoform. |
| Expression | Calretinin is expressed widely amongst inhibitory interneurons in the brain with particularly high expression in the cerebellum and hippocampus. It is also expressed in peripheral tissues such as the testes, lung, pancreas and kidney. |
| Subcellular expression | Calretinin is primarily expressed cytosolically although some nuclear expression has been reported. |
| Processing | Calretinin is not subject to any processing before achieving an active conformation |
| Post translational modifications | Calretinin is subject to phosphorylation on tyrosine 214. |
| Homology (compared to human) | Mouse and rat calretinin show 98.5% and 98.9% homology respectively to human calretinin. Mouse and rat calretinin only show 1 amino acid difference (M271V). |
| Similar proteins | In a BLAST search only Calbindin (58.5% identity, 28kDa) was identified as being a similar protein. |
Storage & Handling
| Storage instructions | -20°C then use reconstitution advice |
| Reconstitution advice | We recommend reconstituting with either:
Take care when opening as the precipitate is extremely light and can easily be lost if disturbed. When reconstituting make sure that the antibody is thoroughly dissolved by pipetting up and down before giving the antibody a brief spin at <10,000g to make sure that all material is recovered and at the bottom of the tube. For more information please see our detailed guide on storing and using your antibody |
| 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) References for Anti-Calretinin antibody ValidAbTM
References are publications that support the biological activity of the product
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Calretinin-Expressing Synapses Show Improved Synaptic Efficacy with Reduced Asynchronous Release during High-Rate Activity.
Zhang C et al (2022) The Journal of neuroscience : the official journal of the Society for Neuroscience 42 : 2729-2742 -
Calretinin positive neurons form an excitatory amplifier network in the spinal cord dorsal horn.
Smith KM et al (2019) eLife 8 : -
Structural and molecular heterogeneity of calretinin-expressing interneurons in the rodent and primate striatum.
Garas FN et al (2018) The Journal of comparative neurology 526 : 877-898 -
Calretinin: from a "simple" Ca(2+) buffer to a multifunctional protein implicated in many biological processes.
Schwaller B (2014) Frontiers in neuroanatomy 8 : 3 -
Calretinin as a marker for cardiac myxoma. Diagnostic and histogenetic considerations.
Terracciano LM et al (2000) American journal of clinical pathology 114 : 754-9
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