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Klymkowsky Lab Methods

Klymkowsky Lab On-line Methods
wax-sectioning and staining
after Fagotto & Gumbiner, 1994

Table of Methods updated - 10 May 2002

Plakoglobin in the epidermis

  
  1. Heat steel embedding dish over flame briefly before adding wax  (this prevents bubbles from forming and allows even cooling of the wax)
  2. Place enough wax to fill embedding dish
  3. Add specimen to dish and orient into desired position
  4. Place embedding ring on the embedding dish (make sure that there is enough wax in the dish so that the embedding ring becomes slightly immersed in the wax - this holds the ring in place)
  5. Add hot wax to the dish until it nearly reaches the top of the embedding ring
  6. Carefully reorient the specimen if it has moved
  7. Cool on cool surface, overnight
  8. Remove dish and section
  1. Fix samples and then into 100% Methanol (for storage at -20*C
  2. If specimens need to be bleached, bleach in Dentís Bleach for 1/2 hour under white light. Store specimens at -20*C in 100% EtOH
  3. 2X washes with 100% EtOH, 5 minutes each
  4. Add 50% EtOH and 50% Hemo-De, 20 minutes for large specimens
  5. 2X washes with 100% Hemo-De
  6. Heat to 58°C in oven, 10 minutes
  7. Vacuum to remove bubbles: - Close purge - Open vacuum  Turn on pump 
       -Vacuum up to 19-20 inches of mercury  Close vacuum knob, as you do turn off pump
  8. Add melted wax (Oxford Paraplast tissue embedding medium;  pelleted form melting point 56°C,  Cat # 8889-501006) in a 1:1 ratio with Hemo-De, one hour
  9. Replace with 100% wax, 2 hours at 58°C
  10. Replace with 100% wax, overnight at 58°C *All times are approximate.  Large samples require longer time. 
  11. section
Table of Methods