[Histonet] non-xylene processing

[ewj <@t> pigsqq.org]

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