Rapid Processing of Fresh Tissue into Paraffin
Fresh tissue <2mm thick can be processed to a block
ready to section in 2 hours. In the same period of time a needle
biopsy will yield a stained cover-slipped slide for diagnosis.
A. Formalin Fixation
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Fixative:
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10% neutral buffered
formalin
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Containers:
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Tissue placed in standard
cassettes - the number of cassettes processed will determine
fixative volume and container size.
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Water Loads:
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500ml recirculated at
the rear of the MW + 250ml on the left to create a cold spot.
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Protocol:
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Tissues <2mm
thick
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Tissue - Needle
Biopsy
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TR: 40°C
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TR: 40°C
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40 min. at
450W
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3 min. at 450W
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10 min. at 650W
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(under vacuum-
20" Hg)
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B. Dehydration/Intermedium: These are straightforward
steps that are done with a temperature restriction (TR). Rinse
in fresh solution prior to each step. The dehydration and intermedium
steps are described below.
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Reagent:
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Ethanol
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Containers:
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Tissue placed in standard cassettes - the number
of cassettes processed will determine fixative volume and container
size.
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Water Loads:
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500ml recirculated at the rear of the MW + 250ml
on the left to create a cold spot.
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Protocol:
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Ethanol Dehydration
for tissues <2mm in thickness - TR=40°C
Rinse in 70% ethanol
70% ethanol 5 min. at 450W
95% ethanol 5 min. at 450W
3 x 100% ethanol 5 min. each at 450W
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NOTE:
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For a needle biopsy
the same steps are done except the time is reduced to 40 sec.
for each step.
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Reagent:
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Xylene
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Containers:
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Tissue placed in standard
cassettes - the number of cassettes processed will determine
fixative volume and container size.
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Water Loads:
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500ml recirculated at
the rear of the MW +250ml on the left to create a cold spot.
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Protocol:
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Inter medium for tissues <2mm
in thickness
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Fresh xylene for 5 min. at 450W
and 5 min. at 550W
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(TR=45°C)
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Fresh xylene for 10min. at 650W
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(TR = 65°C)
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NOTE:
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For a needle biopsy, the same steps are done,
except the time is reduced to 2.5 min. for each step
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C. Paraffin Step:
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Reagent:
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Paraplast® Plus
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Containers:
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Tissue placed in standard
cassettes - the number of cassettes processed will determine
fixative volume and container size.
|
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Water Loads:
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500ml recirculated at
the rear of the MW + 250ml on the left to create a cold spot.
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Protocol:
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Paraffin infiltration for tissues
<2mm in thickness - TR = 63°C
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Rinse in warm (60°) paraffin
Fresh paraffin for 10 min. at 650W - 2 vacuum cycles*
Fresh paraffin for 10 min. at 650W - 2 vacuum cycles*
Fresh paraffin for 10 min. at 650W - 2 vacuum cycles*
*cycle= 2 min. vacuum +2 min. no vacuum +2 min. vacuum +4 min.
no vacuum
(vacuum= 20" Hg; 500 torr)
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NOTE:
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For a needle biopsy, the same steps are done,
except the time is reduced to 6 min. for each step. (the 4 min.
no vacuum component of the 10 min. cycle is dropped)
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Conclusions:
1. Microwave-assisted processing of tissues, from fixation
through paraffin, can save time and produce better results, when
the proper microwave technology is used to get the job done. The
ability to control temperatures by a number of different parameters
(variable wattage, temperature restrictive temperature probe and
water recirculation) make it possible to achieve results that
were not formerly available from other microwave processing systems
or protocols. The technology makes it possible to employ any of
the routinely used reagents for paraffin processing.
2. Variable wattage eliminates the problems reported
when tissues are transferred between solutions having different
microwave transparencies.
go to PELCO BioWave® Pro Microwave Processor
go to Microwave Processor Equipment and Accessories