Re: [Histonet] non-xylene processing

[ewj <@t> pigsqq.org]

We used Dawn until we ran out.   Dawn can work.  It is hard to get here.

We used calgon with the dawn because the water is so hard.


>  -------Original Message-------
>  From: Michelle Moore <mmooreht <@t> yahoo.com>
>  To: ewj <@t> pigsqq.org <ewj <@t> pigsqq.org>, histonet <@t> lists.utsouthwestern.edu <histonet <@t> lists.utsouthwestern.edu>
>  Subject: Re: [Histonet] non-xylene processing
>  Sent: Jul 18 '13 03:27
>  
>  We also de-wax slides in a similar way as the procedure listed below. We
>  also use a waterbath and dish soap (Dawn)/rinse agent method. (Rinse agent
>  (Cascade) helps with water tension.) The key is water temp. it has to be
>  over 90 C in order for it to work. Obviously if you de-wax non-chemical you
>  do not have the alcohols on the stain line either = huge cost savings all
>  the way around, not to mention the healthier work area!
>  Michelle Moore
>  SRMC
>  St. Thomas, USVI
>  
>  
>  FROM: "ewj <@t> pigsqq.org" <ewj <@t> pigsqq.org>
>  TO: histonet <@t> lists.utsouthwestern.edu
>  SENT: Wednesday, July 17, 2013 1:01 PM
>  SUBJECT: [Histonet] non-xylene processing
>  
>  
>  We now have a no-xylene method start to finish
>  
>  We have a Midea (Mei Di) 800W manual dial microwave oven
>  that we have rigged with a K-type metal shielded thermocouple
>  that goes in through a slightly enlarged vent hole in the internal
>  guide housing.  The internal length of the thermocouple wire is
>  wrapped with heavy duty aluminum foil to block microwaves from
>  creating silly currents in the thermocouple wire.  PT100 RTD's
>  are great for water baths but microwaves zap them in seconds.
>  
>  The hysteresis temp controller switches a 12v relay which drives
>  a 10A 240V relay that supplies power to a plain socket for the microwave
>  oven.
>  The microwave oven is a manual dial type so just cutting the power
>  turns it off and on quite effectively.  A low power (% time) setting
>  is enough.  High settings give too much overshoot.
>  The 12V DC  circuit allows us to eliminate problems with the
>  back EMF from the MW oven and 240V relay.  We
>  have a diode that blocks the DC back EMF.
>  
>  Cut in well fixed tissues - fixed in formalin or Bouins.
>  Microwave Cassettes in 100% Isopropanol at 60C - 10 min
>  Repeat with fresh IsopOH another 10 min.
>  
>  Put cassettes in a fired clay earthenware pot -
>  Pour in paraffin heated  to 75C in a metal teapot.
>  Microwave 10 min at 80C
>  Pour out paraffin into open metal pot.
>  Repeat above 10 min.
>  Pour out paraffin to metal pot.
>  
>  Take cassettes to the embedding station,
>  Embed, ice, trim, section.
>  Bake 20 min on slide warmer at 60C
>  
>  We dewax with 95C tap water heated in a tank.
>  A PT100 RTD drives a hysteresis controller.
>  Also we use 12 V relay switched by the controller to drive the 240V
>  relay that switches the heating element on and off.
>  
>  We have an electric solenoid valve and a waterproof switch to  control the
>  water flow when we need the hot water.
>  
>  The hot water flows into a 1 L pot with silicone rubber handles.
>  We add 20 gm of a dishwasher detergent powder made in Shanghai.
>  (It's similar to Cascade.  We dont like Finish very much as sold in China).
>  
>  We put in the slide racks after the water and detergent are in the pot.
>  We treat for one minute, remove the slides, pour out the
>  water and repeat for another 1 minute soak.
>  Next comes a 30 sec soak in clear hot tap water
>  and a 1 min rinse in plain running tap water.
>  
>  Then
>  0.5% Periodic acid 10 min
>  Tap water rinse 1 min
>  Distilled water 1 min
>  Harris Hematoxylin 4 min
>  Rinse tap water 1 min
>  1% HCl in 70% EtOH 30 sec
>  Rinse and blue in tap water 1 min
>  Eosin/Biebrich Scarlet 30 sec
>  Rinse tap water 1 min
>  Rinse Distilled Water 30 sec
>  Dry with hair dryer
>  Coverslip.
>  
>  Our mounting medium contains xylene as a clearing  agent/diluent.
>  That's the only xylene in the normal runs.
>  
>  We like this method because it is quick, environmentally friendly, and
>  people friendly
>  and it produces very lovely slides that we can read and photograph for our
>  reports.
>  
>  A major part of our work is diagnosis of pig diseases, so the ability to
>  give same-day
>  histopath results on formalin fixed samples is valuable to our customers.
>  
>  We do still use xylene to dewax formalin fixed paraffin block sections that
>  are used for PCR presently,
>  but we think we can use hot soapy water for that also with some larger
>  tubes.
>  
>  We heat the teapot on an induction stove.  We have that rigged for thermal
>  control also
>  and a PT100 in through the lid in the teapot.  It is much trickier
>  controlling the induction
>  stove, because they have many built-in safety features and you can't just
>  turn one off and
>  on again at the mains outlet.  What you have to do is  solder in leads to a
>  relay across the
>  on-off push button, and rig the relay for momentary contact by including a
>  capacitor that is charged
>  for "on" and discharged for "off", with a momentary contact at each time
>  faking a key press.
>  This lets us heat our paraffin teapot accurately with induction stove
>  efficiency and automatic temp control.
>  
>  E. Wayne Johnson
>  Enable Ag Tech
>  Beijing
>  
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