List of equipment, chemicals and solutions necessary for cryo-sectioning and EM immunocytochemistry

This is a basic list of equipment and supplies you will need to prepare cryosections of biological material and label them with antibodies. We assume that you have the biological material and the primary antibodies.

List 1: Equipment

  • Transmission Electron Microscope (no special attachments needed).
  • Glass knife maker.
  • Ultra-microtome equipped with cryo-attachment.
  • Anti-static device for ultramicrotome.
  • Vacuum evaporator.
  • Glass strips (for making glass knives).
  • Glass Knife Storage Boxes.
  • Diamond Cryo-Knife.
  • Tungsten wire (for coating newly made glass knives).
  • Aluminium specimen mounting pins.
  • Eyelashes mounted on long wooden sticks.
  • Loops.
  • Fine Paint Brush.
  • Plastic Petri Dishes (3.5 mm diameter).
  • Fine Forceps (e.g. Dumont Biologie #5 or #7).
  • EM Specimen Grids (Hexagonal 100 mesh grids which have been formvar and carbon coated and which have preferably been glow discharged. Copper, nickel, gold and paladium grids can all be used).
  • Grid Boxes.
  • Plastic Wash Bottles.
  • Filter Paper.
  • Stereo Microscope with Light Source.
  • Scalpel blades and Handles.
  • Ice Bucket.
  • Liquid Nitrogen Thermos Flasks with open neck.
  • Liquid Nitrogen Storage Tank (for specimen storage).
  • Parafilm.
  • Eppendorf Tubes and Tube Racks.
  • Plastic Pasteur Pippettes (or Glass).
  • Scissors.
  • Razor Blades.
  • Marker Pens.
  • Adhesive Tape.
  • Timers.
  • Small Beakers.
  • "Dust Off" Aerosol Spray.
  • Kleenex Tissues.

List 2: Chemicals and Solutions

  • Glutaraldehyde (0.5% in a suitable buffer).
  • Formaldehyde (4% - 8% in suitable buffer).
  • Phosphate Buffered Saline (PBS).
  • 2.1M and 2.3 M Sucrose in PBS.
  • PVP-Sucrose in PBS.
  • Distilled Water.
  • Fetal Calf Serum (10% in PBS).
  • 0.12% Glycine in PBS.
  • Secondary antibodies and protein A-gold.
  • 3% Uranyl Acetate in Water.
  • 2% Methyl Cellulose in Water (Use 25 cps viscosity methyl cellulose. It dissolves better in cold water so mix the powder with hot water and leave to dissolve at 4*C for about 3 days. Centrifuge the solution before use in a Ti-70 rotor at 60000 rpm for 2 hours).
  • 2% Poly(vinyl)alcohol in water.
  • Soap Solution for cleaning glass strips.
  • Alcohol for cleaning other equipment (forceps, loops, eyelashes).

Notes

  • Loops are made from copper or platinum wire 0.2 mm diameter and mounted on wooden or plastic sticks, or Eppendorf tips. Two sizes of loop will be required.
    1. Small loops (1-2 mm diameter) for section retrieval
    2. Large loops (3.5 mm diameter) for the final drying step. (These can be made by wrapping the wire around a 3.5 mm drill bit). Make many.
  • Rabbit IgGs which recognize mouse or rat IgGs or IgMs (if the primary antibodies are mouse monoclonals - anti Rat antibodies if the primaries are rat monoclonals etc.) will be required if protein A-gold is to be used to detect all antibodies. This is an economical and simple alternative to purchasing immunoglobulins coupled to colloidal gold.
    Protein A-gold preparations with different particle size are required if double labeling is to be performed.
Ahmanson Center for
Advanced Electron Microscopy and Imaging