Materials and Reagents

1. Paraformaldehyde (PFA)
   It can be ordered as 16% PFA from Electron Microscopy Sciences (catalog number: 15710 ), and diluted to 4% before use. However, we usually make the 4% solution from the powder (Sigma-Aldrich, catalog number: 158127 ). Here is how to make 4% PFA:
   1. In a 500 flask, weigh 4 g PFA in 80 ml PBS.
      
   2. Stir and heat the mixture on a magnetic heater. Keep the temperature below 60 °C during the entire process to avoid breakdown of the polymers.
      
   3. When temperature is close to 60 °C, add a few drops of 10 N sodium hydroxide. The mixture will gradually become clear.
      
   4. Filter the solution into a new flask and cool down to room temperature.
      
   5. Neutralize with hydrochloric acid to pH 7.
      
   6. Add PBS to 100 ml.
      
2. O.C.T. (TFM Tissue Freezing Medium, TFM-5, clear color)
   
3. Phosphate buffered saline (PBS)
   
4. Goat serum
   
5. Triton X-100
   
6. Secondary antibodies:
   Cy2 (or cy3, or cy5)-conjugated goat-anti-mouse (or rabbit) IgG.
   (Jackson ImmunoResearch, F (ab) 2 fragments of affinity-purified antibody)
   
7. Hoechst33258 (Sigma-Aldrich, catalog number: 861405 )
   
8. Antifade reagent (Life Technologies, Invitrogen™, catalog number: P36930 )
   
9. Ethanol
   
10. 4 N hydrochloric acid
    
11. Sodium hydroxide
    
12. Blocking buffer (see Recipes)
    

Equipment

1. Plastic cryomolds (Ted Pella, catalog number: 27110 )
   
2. Cryostat sectioning machine
   
3. SuperFrost Plus slides (VWR International, catalog number: 48311-703 )
   
4. Dark humidified chamber: Made from a square petri dish containing kimwipes that are soaked in water. Wrap the chamber with aluminum foil to protect the slides from light.
   
5. PAP pen
   
6. 500 flask
   
7. Ice bucket
   
8. Magnetic heater
   

Procedure