Rat brain tissues were embedded in paraffin and sliced into sections (3 μm thick) using a microtome. After deparaffinization, dehydration, antigen retrieval, and blocking, the sections were incubated at 4°C overnight with a rat anti-Hdac2 antibody (1:400, 5113s, Cell Signaling Technology), a rabbit anti-Kv1.2 antibody (1:200, APC-010, Alomone Labs), a murine anti-neuronal nuclei (NeuN) antibody (1:300, 66836-1-Ig, Proteintech), a mouse anti-glial fibrillary acidic protein (GFAP) antibody (1:600, 3670, Cell Signaling Technology), or a mouse antiionized calcium-binding adaptor molecule 1 (Iba1) antibody (1:100, ab283319, Abcam). Subsequently, the sections were washed three times with phosphate-buffered saline (PBS) and incubated at 37°C for 2 hours with a goat anti-rat secondary antibody (1:200, 112-605-003, Jackson ImmunoResearch, West Grove, PA, USA) or a goat anti-rabbit secondary antibody (1:200, Jackson ImmunoResearch) . After washing the sections three times, images were taken using a Leica DMI4000 fluorescence microscope equipped with a DFC365FX camera (Leica, Wetzlar, Germany).
Apc 010
Manufactured by Alomone
Sourced in Israel
APC-010 is a laboratory equipment product from Alomone. It is designed for general laboratory use.
Automatically generated - may contain errors
Lab products found in correlation
Ab283319, by Abcam (1 mentions)
Dfc365 fx camera, by Leica camera (1 mentions)
Goat anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
Dmi4000 fluorescence microscope, by Leica camera (1 mentions)
Fluorescent secondary antibodies, by Rockland Immunochemicals (1 mentions)
Sc 7020, by Santa Cruz Biotechnology (1 mentions)
Odyssey scanner, by LI COR (1 mentions)
2 protocols using apc 010
1
Immunohistochemical Analysis of Rat Brain
2
Immunoprecipitation and Immunoblotting of Hypoxia-Sensitive Kv Channels
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The smooth muscle layer of resistance PA rings was dissected. Total protein extracts and protein concentrations were prepared as previously described (2 (link)).
Immunoprecipitations were performed as follows: Lysates containing 0.5 mg total protein were incubated with 10 μg Kv1.2 (APC-010; Alomone Lab, Israel), Kv1.5 (APC-004), Kv2.1 (APC-012), Kv3.1b (APC-014), or Kv9.3 (HPA014864; Sigma, USA) monoclonal antibody overnight at 4°C, which were then captured by Protein A-Sepharose CL-4B beads (Santa Cruz Biotechnology, USA). The beads were collected by centrifugation at 200 g for 3 min at 4°C followed by 3 washes with lysis buffer. After the final wash, the immunoprecipitates were subjected to western blot analyses.
Immunoblotting was performed to detect phosphotyrosine levels in hypoxia-related Kv channels as described previously (24 (link)). Membranes were immunoblotted using antibodies to p-Tyr (SC-7020, Santa Cruz Biotechnology) at 4°C overnight. The signal intensity of the immunoreactive p-Tyr bands from the membrane protein was normalized to Kv channel expression. Fluorescent secondary antibodies (Rockland, USA) were visualized using an Odyssey scanner (LI-COR, USA).
Immunoprecipitations were performed as follows: Lysates containing 0.5 mg total protein were incubated with 10 μg Kv1.2 (APC-010; Alomone Lab, Israel), Kv1.5 (APC-004), Kv2.1 (APC-012), Kv3.1b (APC-014), or Kv9.3 (HPA014864; Sigma, USA) monoclonal antibody overnight at 4°C, which were then captured by Protein A-Sepharose CL-4B beads (Santa Cruz Biotechnology, USA). The beads were collected by centrifugation at 200 g for 3 min at 4°C followed by 3 washes with lysis buffer. After the final wash, the immunoprecipitates were subjected to western blot analyses.
Immunoblotting was performed to detect phosphotyrosine levels in hypoxia-related Kv channels as described previously (24 (link)). Membranes were immunoblotted using antibodies to p-Tyr (SC-7020, Santa Cruz Biotechnology) at 4°C overnight. The signal intensity of the immunoreactive p-Tyr bands from the membrane protein was normalized to Kv channel expression. Fluorescent secondary antibodies (Rockland, USA) were visualized using an Odyssey scanner (LI-COR, USA).
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