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Immunofluorescence staining of kidney tissue from homozygous ENaC γ -VF mice. (A) Schematic of the tagged ENaC γ subunit produced from the ENaC γ -VF allele, which includes a C-terminal mVenus, 3C protease site, and 3xFLAG tag. (B) Representative immunofluorescence image showing colocalization of mVenus and ENaC γ in kidney tissue. mVenus signal was detected using a FITC-conjugated anti-GFP primary antibody (goat, Rockland, 1:100) and Alexa Fluor 488 donkey anti-goat secondary antibody. Endogenous ENaC γ was detected using a rabbit anti-ENaC γ primary antibody (StressMarq, 1:500) targeting the C-terminus, and Alexa Fluor 555 donkey anti-rabbit secondary antibody. (C) Close-up view highlighting localization of tagged ENaC γ . Anti-GFP, anti-green fluorescent protein; DAPI, 4′,6-diamidino-2-phenylindole; ENaC, epithelial sodium channel; ENaC γ -VF, epithelial sodium channel γ - m Venus FLAG.

Journal: Kidney360

Article Title: Differential Assembly of Native Epithelial Sodium Channel Complexes Across Mouse Epithelial Tissues

doi: 10.34067/KID.0000001261

Figure Lengend Snippet: Immunofluorescence staining of kidney tissue from homozygous ENaC γ -VF mice. (A) Schematic of the tagged ENaC γ subunit produced from the ENaC γ -VF allele, which includes a C-terminal mVenus, 3C protease site, and 3xFLAG tag. (B) Representative immunofluorescence image showing colocalization of mVenus and ENaC γ in kidney tissue. mVenus signal was detected using a FITC-conjugated anti-GFP primary antibody (goat, Rockland, 1:100) and Alexa Fluor 488 donkey anti-goat secondary antibody. Endogenous ENaC γ was detected using a rabbit anti-ENaC γ primary antibody (StressMarq, 1:500) targeting the C-terminus, and Alexa Fluor 555 donkey anti-rabbit secondary antibody. (C) Close-up view highlighting localization of tagged ENaC γ . Anti-GFP, anti-green fluorescent protein; DAPI, 4′,6-diamidino-2-phenylindole; ENaC, epithelial sodium channel; ENaC γ -VF, epithelial sodium channel γ - m Venus FLAG.

Article Snippet: Primary antibodies included FITC-conjugated goat anti-green fluorescent protein (anti-GFP; Rockland, 1:100) for mVenus detection, rabbit anti-rat ENaC γ (Stressmarq, 1:500), mouse anti-Aquaporin-2 (Santa Cruz, 1:50), rabbit anti-ENaC α (Loffing Lab, 1:500), and chicken anti-phosphorylated sodium-chloride cotransporter (Welling Lab, 1:5000).

Techniques: Immunofluorescence, Staining, Tissue, Produced