Live‐Animal Imaging of Renal Function by Multiphoton Microscopy

Kenneth W. Dunn1, Timothy A. Sutton1, Ruben M. Sandoval1

1 Indiana University School of Medicine, Indianapolis, Indiana
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.9
DOI:  10.1002/cpcy.32
Online Posting Date:  January, 2018
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Abstract

Intravital microscopy, microscopy of living animals, is a powerful research technique that combines the resolution and sensitivity found in microscopic studies of cultured cells with the relevance and systemic influences of cells in the context of the intact animal. The power of intravital microscopy has recently been extended with the development of multiphoton fluorescence microscopy systems capable of collecting optical sections from deep within the kidney at subcellular resolution, supporting high‐resolution characterizations of the structure and function of glomeruli, tubules, and vasculature in the living kidney. Fluorescent probes are administered to an anesthetized, surgically prepared animal, followed by image acquisition for up to 3 hr. Images are transferred via a high‐speed network to specialized computer systems for digital image analysis. This general approach can be used with different combinations of fluorescent probes to evaluate processes such as glomerular permeability, proximal tubule endocytosis, microvascular flow, vascular permeability, mitochondrial function, and cellular apoptosis/necrosis. © 2018 by John Wiley & Sons, Inc.

Keywords: fluorescence microscopy; intravital microscopy; in vivo microscopy; multiphoton microscopy

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Glomerular Permeability
  • Basic Protocol 2: Proximal Tubule Endocytosis
  • Basic Protocol 3: Vascular Flow
  • Basic Protocol 4: Vascular Permeability
  • Basic Protocol 5: Mitochondrial Function
  • Basic Protocol 6: Apoptosis
  • Support Protocol 1: Anesthesia and Surgical Creation of a Retroperitoneal Surgical Window for Intravital Imaging
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Glomerular Permeability

  Materials
  • Munich Wistar Frömter rat (not commercially available, but available from the Indiana O'Brien Center for Advanced Microscopic Analysis; http://medicine.iupui.edu/neph/obrien/center)
  • 70,000‐Da dextran–Alexa 488 (see recipe)
  • 40,000‐Da dextran‐rhodamine (see recipe)
  • Isotonic saline (0.9% NaCl), sterile
  • 10 mg/ml Hoechst 33342 (Invitrogen) in sterile distilled H 2O
  • Intravital microscopy system (see Commentary)
  • Additional reagents and equipment for anesthesia and preparation for intravital imaging ( protocol 7Support Protocol) and injection of rodents (Donovan & Brown, )

Basic Protocol 2: Proximal Tubule Endocytosis

  Materials
  • Animal
  • 3,000‐Da dextran–Alexa 488 (see recipe), 10,000‐Da dextran‐rhodamine (see recipe), or fluorescent bovine serum albumin (see recipe)
  • Isotonic saline (0.9% NaCl), sterile
  • Intravital microscopy system (see Commentary)
  • Additional reagents and equipment for anesthesia and preparation for intravital imaging ( protocol 7Support Protocol) and injection of rodents (Donovan & Brown, )

Basic Protocol 3: Vascular Flow

  Materials
  • Animal
  • Fluorescent 500,000‐Da dextran (see recipe) or fluorescent bovine serum albumin (see recipe)
  • Isotonic saline (0.9% NaCl), sterile
  • Intravital microscopy system (see Commentary)
  • Additional reagents and equipment for anesthesia and preparation for intravital imaging ( protocol 7Support Protocol) and injection of rodents (Donovan & Brown, )

Basic Protocol 4: Vascular Permeability

  Materials
  • Animal
  • Fluorescent 500,000‐Da dextran (see recipe)
  • Fluorescent 10,000‐Da dextran, 40,000‐Da dextran, 70,000‐Da dextran, 150,000‐Da dextran (see recipe), or fluorescent‐labeled bovine serum albumin (see recipe): choice of probe will depend upon the intrinsic permeability of the vessel of interest
  • Isotonic saline (0.9% NaCl), sterile
  • Intravital microscopy system (see Commentary)
  • Additional reagents and equipment for anesthesia and preparation for intravital imaging ( protocol 7Support Protocol) and injection of rodents (Donovan & Brown, )

Basic Protocol 5: Mitochondrial Function

  Materials
  • Animal
  • Rhodamine B hexyl ester (see recipe)
  • Isotonic saline (0.9% NaCl), sterile
  • 10 mg/ml Hoechst 33342 solution to label nuclei (see recipe; optional)
  • Intravital microscopy system (see Commentary)
  • Additional reagents and equipment for anesthesia and preparation for intravital imaging ( protocol 7Support Protocol) and injection of rodents (Donovan & Brown, )

Basic Protocol 6: Apoptosis

  Materials
  • Animal
  • 10 mg/ml Hoechst 33342 (Invitrogen)
  • 5 mg/ml propidium iodide (Invitrogen)
  • Isotonic saline (0.9% NaCl), sterile
  • Intravital microscopy system (see Commentary)
  • Additional reagents and equipment for anesthesia and preparation for intravital imaging ( protocol 7Support Protocol) and injection of rodents (Donovan & Brown, )

Support Protocol 1: Anesthesia and Surgical Creation of a Retroperitoneal Surgical Window for Intravital Imaging

  Materials
  • Animal to be imaged
  • 5% (v/v) and 2% (v/v) isoflurane/oxygen mixtures
  • Pentabarbital (optional)
  • Buprenorphine
  • Germicidal soap
  • Isotonic saline (0.9% NaCl), sterile, prewarmed
  • Appropriate probes (see protocols above)
  • Anesthesia induction chamber (Braintree Scientific)
  • Homeothermic table (Braintree Scientific)
  • Electric clippers
  • Rectal probe (Braintree Scientific)
  • Surgical scissors (Braintree Scientific)
  • Vascular catheters (PE‐60 tubing for rats and PE‐50 tubing for mice; Becton Dickinson)
  • Intravital microscopy system (see Commentary)
  • 50‐mm cell culture dish fitted with a number 1‐1/2 coverslip in the bottom (Warner Instruments, cat. no. GWST‐5040)
  • Appropriate temperature‐control devices (e.g., Heated water jacket (Kent Scientific TPZ‐1215VF on a TPZ‐747 Micro‐Temp LT circulation pump and Repti Therm heating pads)
  • Objective heater (OW series heater with TC124 controller, Warner Instruments)
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Figures

Videos

Literature Cited

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