Imaging isolated and in situ bladder arterioles (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Gerard Ahern

Keywords

[‘immunofluorescence’]

Subjects

[‘Imaging’, ‘Immunological techniques’]

Release Date

2017-08-07

Abstract

Imaging isolated and in situ bladder arterioles protocol by the Ahern Group

Procedure

• Bladders are removed from TRPV1-Cre:tomato mice, ventrally opened longitudinally from the bladder neck to the top of the dome.
• Under a dissecting microscope the urothelium is carefully peeled away using iris scissors to expose sub-urothelial arterioles.
• The preparation is placed in 500 μL of physiological buffer containing 140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 1.2 mM CaCl2, 10 mM HEPES, 5 mM glucose, pH=7.3, and anchored by platinum-wire tissue harps.
• If desired individual 3rd order arterioles (~20-40 μm diameter) can be carefully microdissected using iris scissors. Isolated arterioles are placed in a micro-insert chamber (Ibidi Inc., volume 10 μL) containing 5 μL of physiological buffer.
• Stock solutions of capsaicin (1 M and 10 mM in EtOH) and the TRPV1 antagonist, BCTC (2 mM in EtOH) are prepared. From these stocks working solutions of 2 mM (in situ studies) and 10μM (isolated arterioles) capsaicin are prepared. A working solution containing both capsaicin (10μM) and BCTC (2μM) is also prepared.
• tdTomato is excited at 540±12 nm and emitted florescence is passed through a 620±30 nm bandpass filter. Experiments are digitally recorded at a frame rate of at least 1 per second.
• Isolated arteries are challenged with 5 μL of 2x capsaicin (10μM) to generate a final concentration of 5μM capsaicin. Subsequently, 5 μL is withdrawn from the chamber and replaced with 5 μL of capsaicin/BCTC (10 μM and 2μM) solution.
• For in situ preparations a higher capsaicin concentration (~1mM) is required due to the tissue absorption of the drug. 500 μL of 2x (2 mM) capsaicin is added to chamber to yield a final concentration of 1mM capsaicin.
• At the end of the experiment a solution containing KCl (50 mM) is used as a positive control.
• Arterial diameter is measured post-hoc at several locations along the artery using the edge-detection plug-in for ImageJ.

Consortium

GenitoUrinary Development Molecular Anatomy Project (GUDMAP) Consortium