Journal: Journal of visualized experiments : JoVE
Article Title: Streamlined Intravital Imaging Approach for Long-Term Monitoring of Epithelial Tissue Dynamics on an Inverted Confocal Microscope
doi: 10.3791/65529
Figure Lengend Snippet: Materials
Article Snippet: Name Company Catalog Number Comments 3D Printer Qudi Tech i-Fast 3D prints using PLA material 40x 1.25NA silicone objective lens Nikon AxR Laser Scanning Confocal Microscope Nikon Cotton Tipped Swab VWR 76337–046 Cream/ointment application Doxycycline hyclate Sigma-Aldrich D9891 Induces GFP labeling of fibroblast nuclei in Pdgfra-rtTA; pTRE-H2B- GFP mice Flathead Screwdriver (2.5 mm) Affiix insert to microscope stage Flathead Screws x 4 (#6–32) Nikon Screw insert into microscope stage Glass Bottom Culture Dish chemglass Life Sciences CLS-1811–002 Modified by removing walls of dish for use as coverslip disk compatible with live insert; 35 mm wide disk contains 20 mm wide glass coverslip; dish walls were removed by machine shop Heat Plate controller Physitemp TCAT-2LV Animal Temperature Controller - Low Voltage; anal prob attachment for mouse body temperature monitoring Hex Wrench (1.5 mm) For M3 setscrew adjustments Hex Wrench (2.5 mm) Adjust tension on metal ear clip Intravital Imaging Insert Isoflurane Med-Vet International HPA030782–100uL Mouse anesthesia Labeling Tape (or Scotch Tape) VWR 10127–458 Alternative to metal ear clip to immobilize ear to coverslip Metal fastener used as ear clip Mouse: C57BL/6- Pdgfraeml (rtTA)Xsun/J The Jackson Laboratory RRID: IMSR_JAX:034459 Fibrroblast-specific promoter driving doxycycline-inducible rtTA expression Mouse: K14-H2BPAmCherry Courtesy of Dr. Valentina Greco at Yale University Labels epidermal epithelial cell nuclei with mCherry; referred to in text as "K14-H2B-mCherry" Mouse: pTRE-H2B-GFP: STOCK Tg(tetO-HIST1 H2BJ/GFP)47Efu/J The Jackson Laboratory RRID: IMSR_JAX:005104 Labels fibroblast nuclei with GFP when combined with Pdgfra-rtTA and induced with doxycycline Multipurpose Sealing Wrap Glad Enhance mouse warmth Optixcare VWR MSPP-078932779 Eye lubricant Set screws x 3 (M3; 6 mm) Thorlabs SS3M6 Attachment for heatplate module Silicone Immersion Oil Applied to 40x silicone objective Small Animal Heating Plate Physitemp HP-4M Provides heat to animal Somnoflow Low-Flow Electronic Vaporizer Kent Scientific SF-01 Mouse anesthesia Vacuum Grease Flinn Scientific AP1095 Seals coverslip disk to insert Veet hair removal Water circulating heat pad Stryker Medical TP700 for mouse revival post-imaging Open in a separate window Materials To ensure reliable and efficient imaging over a duration of time using this new tool, it is critical to properly size the live imaging insert according to the user’s microscope stage.
Techniques: Microscopy, Cream, Labeling, Modification, Imaging, Expressing
