PBS-perfused livers were isolated and post-fixed in 4% (w/v) paraformaldehyde in PBS overnight at 4°C before preservation in 30% (w/v) sucrose in PBS. Livers were sectioned at a thickness of 50 μm on a freezing–sliding microtome, and sections were stored in cryoprotective medium at −20°C. Free-floating sections were blocked with 5% donkey serum for 1.5 hours before overnight incubation at 4°C with primary antibodies (goat anti-Albumin antibody, Abcam ab19194, 1:100 dilution; rabbit anti-V5 antibody, Cell Signaling Technology, 1:500 dilution). Sections were washed, stained with Alexa Fluor-conjugated secondary antibodies (Thermo Fisher Scientific, 1:250 dilution) in blocking buffer for 2.5 hours at room temperature, mounted, and coverslipped with ProLong Gold (Life Technologies) before imaging on a confocal laser-scanning microscope (Zeiss LSM880).
Rabbit anti v5 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Rabbit anti-V5 antibody is a primary antibody that specifically recognizes the V5 epitope tag, which is commonly used to detect and purify recombinant proteins. This antibody is produced in rabbits and can be used in various immunoassays, such as Western blotting, immunoprecipitation, and immunohistochemistry, to detect and visualize V5-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880, by Zeiss (4 mentions)
Ab19194, by Abcam (2 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Prolong gold, by Thermo Fisher Scientific (2 mentions)
Eclipse ts2 microscope, by Nikon (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
Trans blot turbo mini 0.2 μm pvdf membrane, by Bio-Rad (1 mentions)
Image studio software, by LI COR (1 mentions)
4 protocols using rabbit anti v5 antibody
1
Immunostaining of Perfused Liver Sections
2
Immunostaining of Perfused Liver Sections
3
Validating CFTR Vector Expression
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The presence of the V5 tag in the third generation CFTR vector was validated in cell culture via immunocytochemistry (ICC) prior to delivery to animals. HEK293T cells were transduced with VSV-G pseudotyped CCL-V5-CFTR lentivirus. Samples were fixed in 4% paraformaldehyde for 10 min at room temperature before being washed in PBS. Cells were permeabilised by incubation in 1% bovine serum albumin (BSA) in PBS containing 0.1% Triton-X 100 for 10 min at room temperature. Cells were washed in PBS and blocked with 1% BSA in PBS for 1 h at room temperature. Samples were incubated in rabbit anti-V5 antibody (1:600) (Cat # 13,202, Cell Signalling Technologies, United States) diluted in 1% BSA + PBS +0.1% Tween-20 (PBST) overnight at 4°C. Samples were washed with 0.1% PBST before being incubated in donkey anti-rabbit Alexa Fluor 488 (1:600) (Cat # ab150073, Abcam, United Kingdom) diluted in 1% BSA + 0.1% PBST for 1 h at room temperature. Cells were washed in 0.1% PBST before incubation in DAPI for 5 min followed by rinsing in 0.1% PBST. The presence of V5 immunofluorescence was confirmed using a Nikon Eclipse Ts2 microscope with NIS-Elements imaging software Version 5.20.00.
4
Western Blot Analysis of V5-tagged Proteins
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Samples from solubilization and purification
steps were mixed with 1× NuPAGE LDS sample buffer and 1×
NuPAGE sample reducing agent. The mixture was heated at 50 °C
for 10 min. SDS-polyacrylamide gel electrophoresis was performed using
NuPAGE 4–12%, Bis-Tris gels. The gel was then transferred onto
a Trans-Blot Turbo Mini 0.2 μm PVDF membrane (Bio-Rad). The
membrane was blocked with 5% BSA in TBS-T (TBS + 0.1% Tween-20) for
1 h. Subsequently, the membrane was incubated with rabbit anti-V5
antibody (1:1000, Cell Signaling Technologies, D3H8Q) for 2 h at RT.
Antibody binding was detected using IRDye 680RD-conjugated Goat Anti-Rabbit
IgG H&L preabsorbed (1:10,000, Abcam, ab216777) for 1 h at RT.
The protein bands were visualized using the Odyssey CLx Imaging System
and quantified using ImageStudio software (Li-Cor).
steps were mixed with 1× NuPAGE LDS sample buffer and 1×
NuPAGE sample reducing agent. The mixture was heated at 50 °C
for 10 min. SDS-polyacrylamide gel electrophoresis was performed using
NuPAGE 4–12%, Bis-Tris gels. The gel was then transferred onto
a Trans-Blot Turbo Mini 0.2 μm PVDF membrane (Bio-Rad). The
membrane was blocked with 5% BSA in TBS-T (TBS + 0.1% Tween-20) for
1 h. Subsequently, the membrane was incubated with rabbit anti-V5
antibody (1:1000, Cell Signaling Technologies, D3H8Q) for 2 h at RT.
Antibody binding was detected using IRDye 680RD-conjugated Goat Anti-Rabbit
IgG H&L preabsorbed (1:10,000, Abcam, ab216777) for 1 h at RT.
The protein bands were visualized using the Odyssey CLx Imaging System
and quantified using ImageStudio software (Li-Cor).
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