Histology Microwave Protocol; fresh tissue into paraffin

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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

Fixative:	10% neutral buffered formalin
Containers:	Tissue placed in standard cassettes - the number of cassettes processed will determine fixative volume and container size.
Water Loads:	500ml recirculated at the rear of the MW + 250ml on the left to create a cold spot.

Protocol:	Tissues <2mm thick	Tissue - Needle Biopsy
	TR: 40°C	TR: 40°C
	40 min. at 450W	3 min. at 450W
	10 min. at 650W	(under vacuum- 20" Hg)

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.

Reagent:	Ethanol
Containers:	Tissue placed in standard cassettes - the number of cassettes processed will determine fixative volume and container size.
Water Loads:	500ml recirculated at the rear of the MW + 250ml on the left to create a cold spot.

Protocol:	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
NOTE:	For a needle biopsy the same steps are done except the time is reduced to 40 sec. for each step.
Reagent:	Xylene
Containers:	Tissue placed in standard cassettes - the number of cassettes processed will determine fixative volume and container size.
Water Loads:	500ml recirculated at the rear of the MW +250ml on the left to create a cold spot.

Protocol:	Inter medium for tissues <2mm in thickness
	Fresh xylene for 5 min. at 450W and 5 min. at 550W	(TR=45°C)
	Fresh xylene for 10min. at 650W	(TR = 65°C)
NOTE:	For a needle biopsy, the same steps are done, except the time is reduced to 2.5 min. for each step

C. Paraffin Step:

Reagent:	Paraplast® Plus
Containers:	Tissue placed in standard cassettes - the number of cassettes processed will determine fixative volume and container size.
Water Loads:	500ml recirculated at the rear of the MW + 250ml on the left to create a cold spot.

Protocol:	Paraffin infiltration for tissues <2mm in thickness - TR = 63°C
	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)

NOTE:	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)

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.

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