Journal: The Journal of Veterinary Medical Science

Article Title: Expression of Adrenergic and Cholinergic Receptors in Murine Renal Intercalated Cells

doi: 10.1292/jvms.14-0315

Figure Lengend Snippet: List of aquaporin and neurotransmitter receptor primers

Article Snippet: Membranes were incubated with diluted primary antibodies including rabbit anti-AQP6 polyclonal antibody (pAb, #AQP-006, Alomone Labs, Jerusalem, Israel), rabbit anti-AQP2 pAb (#AQP-002, Alomone labs), rabbit anti-ADR b2 pAb (#Sc-569, Santa Cruz, TX, U.S.A.) and rabbit anti-mAChR m1 pAb (#010, Alomone Labs) for 2 hr at room temperature or at 4°C overnight.

Techniques:

Journal: The Journal of Veterinary Medical Science

Article Title: Expression of Adrenergic and Cholinergic Receptors in Murine Renal Intercalated Cells

doi: 10.1292/jvms.14-0315

Figure Lengend Snippet: Characterization of M-1 cellular expression of aquaporin A) Characterization of AQP6 mRNA and protein expression in M-1 cells by RT-PCR (upper column) and immunoblot (middle column), respectively. An AQP6 specific band (arrowhead) was amplified from M-1 cellular total RNA (lane 1), M-1 cellular genomic DNA (gDNA, lane 3) and normal mouse kidney total RNA (MK cDNA, lane 4). No positive band was observed in RTase (−) negative control lane (lane 2). Immunoblot demonstrating the AQP6 specific band (arrowhead) and glycosylated band (double arrowhead) in total membrane protein extracted from M-1 cells (lane 1) and murine kidney (MK, lane 2). B) Characterization of AQP2 mRNA and protein expression in M-1 cells by RT-PCR (upper column) and immunoblot (middle column), respectively. AQP2 mRNA was not amplified from M-1 cellular total RNA (lane 1), although it was observed in M-1 gDNA (lane 3) and MK cDNA (lane 4). Similarly, AQP2 protein was not observed in total membrane protein isolated from M-1 cells (lane 1), although it was observed in total membrane protein isolated from the MK (lane 2). An arrowhead and a double arrow indicate the unglycosylated and glycosylated bands, respectively. Immunofluorescence showed the M-1 cells were positive to the AQP6 antibody (A, lower column), but negative to AQP2 antibody (B, lower column). Bar=50 µ m.

Article Snippet: Membranes were incubated with diluted primary antibodies including rabbit anti-AQP6 polyclonal antibody (pAb, #AQP-006, Alomone Labs, Jerusalem, Israel), rabbit anti-AQP2 pAb (#AQP-002, Alomone labs), rabbit anti-ADR b2 pAb (#Sc-569, Santa Cruz, TX, U.S.A.) and rabbit anti-mAChR m1 pAb (#010, Alomone Labs) for 2 hr at room temperature or at 4°C overnight.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Amplification, Negative Control, Isolation, Immunofluorescence

Journal: The Journal of Veterinary Medical Science

Article Title: Expression of Adrenergic and Cholinergic Receptors in Murine Renal Intercalated Cells

doi: 10.1292/jvms.14-0315

Figure Lengend Snippet: Expression of adrenergic receptors in M-1 cells and histological localization of the β2 receptor in the mouse kidney. A) RT-PCR (upper column) of ADRs in M-1 total cellular RNA (cDNA, lane 1) and genomic DNA (gDNA, lane 3). The α2 (second row) and β2 (third row) ADRs were amplified from M-1 total cellular RNA, but the α1 (first row) and β3 (fourth row) ADRs were not. No positive band was observed in RTase (−) negative control lane (lane 2). Immunoblot of the β2 receptor (lower column) shows a specific 60–75 kDa band (arrowhead). B) and C) Immunohistochemistry of the β2 receptor (B) and AQP6 (C) in the mouse renal cortex (arrows indicate the same points on both images). Arrows indicate AQP6-positive, β2-positive cells, and arrowheads indicate AQP6-positive, β2-negative cells in the CCDs. D) and E) Immunohistochemistry of the β2 receptor (D) and AQP6 (E) in the outer medulla. β2 receptor immunoreactivities were observed to the straight segment of proximal tubules, but not in the collecting ducts. Arrowheads (AQP6 positive cells) indicate the same points on both images. G: glomerulus. Bar=20 µ m (B and C) and 100 µ m (D and E).

Article Snippet: Membranes were incubated with diluted primary antibodies including rabbit anti-AQP6 polyclonal antibody (pAb, #AQP-006, Alomone Labs, Jerusalem, Israel), rabbit anti-AQP2 pAb (#AQP-002, Alomone labs), rabbit anti-ADR b2 pAb (#Sc-569, Santa Cruz, TX, U.S.A.) and rabbit anti-mAChR m1 pAb (#010, Alomone Labs) for 2 hr at room temperature or at 4°C overnight.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control, Western Blot, Immunohistochemistry

Journal: The Journal of Veterinary Medical Science

Article Title: Expression of Adrenergic and Cholinergic Receptors in Murine Renal Intercalated Cells

doi: 10.1292/jvms.14-0315

Figure Lengend Snippet: Expression of mAChRs in M-1 cells and immunohistochemical localization of the m1 ADR in the mouse kidney. A) RT-PCR (upper column) of m1, m3, m4 and m5 receptor subtypes in M-1 total cellular RNA (lane 1, arrowheads). Genomic DNA was used as a control (lane 3). No positive band was observed in RTase (−) negative control lane (lane 2). Immunoblot (lower column) demonstrating the presence of the m1 receptor subtype in the M-1 total membrane protein fraction. The most intense band is observed at 55 to 70 kDa (arrowhead). B) to E) Immunohistochemistry demonstrating the presence of the m1 mAChR (B and D) and AQP6 (C and E) in the cortex (B and C) and renal outer medulla (D and E). Arrows indicate AQP6-positive, m1-positive cells. Arrowheads indicate AQP6-positive, m1-negative cells. Reactions of m1 antibody were stronger to the medullary collecting ducts (B) than to the CCDs (D). Bar=20 µ m.

Article Snippet: Membranes were incubated with diluted primary antibodies including rabbit anti-AQP6 polyclonal antibody (pAb, #AQP-006, Alomone Labs, Jerusalem, Israel), rabbit anti-AQP2 pAb (#AQP-002, Alomone labs), rabbit anti-ADR b2 pAb (#Sc-569, Santa Cruz, TX, U.S.A.) and rabbit anti-mAChR m1 pAb (#010, Alomone Labs) for 2 hr at room temperature or at 4°C overnight.

Techniques: Expressing, Immunohistochemical staining, Reverse Transcription Polymerase Chain Reaction, Negative Control, Western Blot, Immunohistochemistry

Journal: The Journal of Veterinary Medical Science

Article Title: Expression of Adrenergic and Cholinergic Receptors in Murine Renal Intercalated Cells

doi: 10.1292/jvms.14-0315

Figure Lengend Snippet: Dual staining of AChE and AQP6 in CCDs. A) AChE was localized to the glomerulus (G) and the nerve fibers along the interlobular arterioles (arrows) B) AChE in CCD cells was stained with the modified Karnovsky-Roots method (arrows). C) AQP6 in the mouse renal cortex was stained by immunohistochemistry (arrowheads). AChE and AQP6 staining did not overlap in the CCDs. The arrows and arrowheads indicate identical cells in panels A and B. Bar=20 µ m.

Article Snippet: Membranes were incubated with diluted primary antibodies including rabbit anti-AQP6 polyclonal antibody (pAb, #AQP-006, Alomone Labs, Jerusalem, Israel), rabbit anti-AQP2 pAb (#AQP-002, Alomone labs), rabbit anti-ADR b2 pAb (#Sc-569, Santa Cruz, TX, U.S.A.) and rabbit anti-mAChR m1 pAb (#010, Alomone Labs) for 2 hr at room temperature or at 4°C overnight.

Techniques: Staining, Modification, Immunohistochemistry