Whole-mount IHC was performed as described previously (30 (link), 68 (link)). The following primary antibodies were used: (a) anti-HCN4 (Abcam, ab66501, 1:200 dilution), a polyclonal antibody raised against rat HCN4, and (b) anti-ACI (Santa Cruz Biotechnology Inc., sc-365350, 1:100 dilution), a monoclonal antibody raised against mouse ACI. SAN tissue was washed with PBS (3 × 10 minutes) and then incubated with anti-rat and anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories, 1:1,000 dilution) for 4 hours at room temperature in the dark. It was then washed in PBS (3 × 10 minutes) and incubated for 2 hours with 20% DMSO diluted in PBS. Coverslips were mounted on the slides with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific). The slides were sequentially imaged using a Zeiss 900 confocal laser-scanning microscope equipped with an Airyscan detector module, a Plan-Apo 63× 1.4 NA oil-immersion objective, and 488/561 lasers. Imaris software (Bitplane) was used to perform 3D reconstructions.
Anti rat and anti mouse secondary antibodies
Manufactured by Jackson ImmunoResearch
Sourced in United States
Anti-rat and anti-mouse secondary antibodies are laboratory reagents used to detect the presence of rat or mouse primary antibodies in various immunoassays, such as Western blotting, ELISA, and immunohistochemistry. These secondary antibodies are labeled with reporter molecules, such as enzymes or fluorescent dyes, to enable visualization and quantification of the target primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Ab66501, by Abcam (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Plan apo 63 1.4 n a, by Zeiss (1 mentions)
Benfotiamine, by Merck Group (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Thiamine, by Merck Group (1 mentions)
Primary antibody rat anti brdu, by Bio-Rad (1 mentions)
2 protocols using anti rat and anti mouse secondary antibodies
1
Whole-mount IHC for HCN4 and ACI
2
Preparation of Thiamine, Benfotiamine, and BrdU Solutions
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Thiamine and benfotiamine were from Sigma-Aldrich NV/SA (Diegem, Belgium).
Thiamine (1.7 g/L or 5 mM) was dissolved in tap water and pH was adjusted to 7 with NaOH.
benfotiamine (1.7 g/L or 3.7 mM) was dissolved in alkalinized tap water and pH was adjusted to 7 with HCl. Bromodeoxyuridine (BrdU, Sigma-Aldrich) was dissolved in 0.9% NaCl and 0.007M NaOH. Primary antibody rat anti-BrdU (1:500, AbD Serotec, Raleigh, NC, USA), anti-rat and anti-mouse secondary antibodies (1:500, Jackson ImmunoResearch, Europe Ltd, Suffolk, U.K.) were used.
Thiamine (1.7 g/L or 5 mM) was dissolved in tap water and pH was adjusted to 7 with NaOH.
benfotiamine (1.7 g/L or 3.7 mM) was dissolved in alkalinized tap water and pH was adjusted to 7 with HCl. Bromodeoxyuridine (BrdU, Sigma-Aldrich) was dissolved in 0.9% NaCl and 0.007M NaOH. Primary antibody rat anti-BrdU (1:500, AbD Serotec, Raleigh, NC, USA), anti-rat and anti-mouse secondary antibodies (1:500, Jackson ImmunoResearch, Europe Ltd, Suffolk, U.K.) were used.
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