PELCO BioWave® Paraffin Tissue Processing Protocol

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Paraffin Tissue Processing (2mm or less) with the PELCO BioWave^® Pro - Protocol
Step#	Description	User Prompt (on/off)	Time (hr:min:sec)	Power (Watts)	Temp. (°C)	Load Cooler (off/auto/on)	Vacuum/ Bubble Pump (off/bub/ vac cycle/ vac on)	SteadyTemp Pump (on/off)	SteadyTemp temp (°C)
1	70% Ethanol Dehydration	On	0:7:0	100	40	Auto	Off	Off	30
2	95% Ethanol Dehydration	On	0:5:0	100	40	Auto	Off	Off	30
3	100% Ethanol Dehydration	On	0:5:0	100	40	Auto	Off	Off	30
4	100% Ethanol Dehydration	On	0:5:0	100	40	Auto	Off	Off	30
5	100% Xylene	On	0:10:0	250	40	Auto	Off	Off	30
6	100% Xylene	On	0:10:0	650	60	Auto	Off	Off	30
7	100% Paraffin or 1:1 with Xylene	On	0:10:0	650	70	Auto	Vacuum Cycle	Off	30
8	100% Paraffin	On	0:10:0	650	70	Auto	Vacuum Cycle	Off	30
9	100% Paraffin	On	0:10:0	650	70	Auto	Vacuum Cycle	Off	30
The times and the number of reagent changes are based on processing tissues that are 2mm or less in thickness. Thicker tissues will require longer times and perhaps added dehydration and clearing steps. Formalin fixation in the microwave is done with a different setup. The second xylene step (#6) is designed to heat the samples to the same temperature as the paraffin. Paraffin is transparent (does not heat) to microwave energy. A paraffin heating tile is used to maintain the temperature during processing. True wattage, not pulsed or percent power makes the use of xylene routine. Substitutes can be used if desired (add steps to insure clearing).

Figure 4A-F. Figures A-C are of normal mouse liver benched fixed in 10% NBF for 24 hours. Figures D-F are of normal mouse liver fixed in 10% NBF for 20 minutes utilizing microwave radiation. All tissues were prepared for fixation identically and cut to 2mm prior to fixation. Figures A and D are corresponding Hematoxylin and Eosin stained sections and figures B and E are corresponding Vimentin IHC stained sections. Figures C and F are corresponding EM sections demonstrating complimentary ultra-structure. Images are from Dr. Jose Galvez, Center for Comparative Medicine and Department of Medical Pathology, University of California, Davis, CA.

Figure 5. Tissues formalin fixed and paraffin processed by the protocols described in Galvez et al. 2006. Mouse mammary tumor virus induced mammary carcinoma (A, B). Note the mitotic figures (arrows) in B. Mouse esophagus with clearly identifiable muscle striations (*) (C). Mouse uterus stained with mouse anti-estrogen (D). (Center for Comparative Medicine and Department of Pathology, Univ. of Calif., Davis)

Special Stains

Mucin – Alcian Blue pH 2.5 - Microwave Procedure¹

Notes:

Place temperature probe in ColdSpot™ port when Temperature Restriction
(TR) is "None" and set TR to >50ºC
Place sample container on ColdSpot™
Check wattage setting
LED above wattage setting indicator will be RED when the magnetron is on
When a TR is set the LED will be RED until the TR is reached then it will turn GREEN (magnetron off) and cycle between RED and GREEN for the remaining time
Place temperature probe in sample container and set TR

Mucin – Alcian Blue pH 2.5 - Microwave Procedure 1

Step	Container Reagent	Wattage Setting	Temperature Restriction	Time/Pad#
1	50ml Coplin Jar xylene Note #2	165W - #1 Note #3	None Note #1	4 min. - #8 Note #4
2	50ml Coplin Jar 95-100% ETOH Note #2	165W - #1 Note #3	None Note #1	1 min. - #3 Note #4
3	Wash in tap water to clear	BENCH STEP
4	50ml Coplin Jar 3% Acetic Acid Notes #2 & #6	165W - #1 Note #3	TR 45°C Note #5	30 sec. - #1 Note #4
5	50ml Coplin Jar Alcian Blue Sol. Notes #2 & #6	165W - #1 Note #3	TR 45°C Note #5	1 min. 30 sec. Note #4
6	Wash in DI water 3 changes	BENCH STEP
7	50ml Coplin Jar Nuclear Fast Red* Notes #2 & #6	165W - #1 Note #3	TR 45°C Note #5	1 min. 30 sec. Note #4
8	Wash in DI water 3 changes	BENCH STEP
9	Dehydrate through ETOH's, clear and mount	BENCH STEP
NOTES Steps #4, #5 and #7 do not require reaching 45ºC

*Or Brazilliant, Anatech

¹Requires a microwave with continuous variable wattage and a ColdSpot™ - Recommended models are a PELCO BioWave^®

REFERENCES:
Carson,F.L., Histotechnology: A Self-Instructional Text, ASCP Press, 1990, pg 130 - 131
Sheehan and Hrapchak, Theory and Practice of Histotechnology, 2nd ed.1980, Mosby
Preece, A Manual for Histologic Technicians, 3rd ed.,1972, Little, Brown & Co.

RESULTS:
Exclusively acid mucosubstances - blue
Nuclei - pale red.

Fungus & Pneumocystis – Grocott's Methenamine Silver Nitrate - Microwave Procedure¹

Notes:

Place temperature probe in ColdSpot™ port when Temperature Restriction (TR) is "None" and set TR to >50ºC
Place sample container on ColdSpot™
Check wattage setting
LED above wattage setting indicator will be RED when the magnetron is on
When a TR is set the LED will be RED until the TR is reached then it will turn GREEN (magnetron off) and cycle between RED and GREEN for the remaining time
Place temperature probe in sample container and set TR

Grocott’s Methenamine Silver Nitrate - Microwave Procedure

Grocott’s Methenamine Silver Nitrate - Microwave Procedure 2

Step	Container Reagent	Wattage Setting	Temperature Restriction	Time/Pad#
1	50ml Coplin Jar xylene Note #2	165W - #1 Note #3	None Note #1	4 min. - #8 Note #4
2	50ml Coplin Jar 95-100% ETOH Note #2	165W - #1 Note #3	None Note #1	1 min. - #3 Note #4
3	Wash in tap water to clear	BENCH STEP
4	50ml Coplin Jar 5% Chromic Acid Notes #2 & #6	502W - #3 Note #3	TR 60°C Note #5	6 min. - #0 Note #4
5	Wash in tap water	BENCH STEP
6	50ml Coplin Jar sodium bisulfite sol.	BENCH STEP		1 min.
7	Wash in tap then 3-4 times in DI water	BENCH STEP
8	50ml Coplin Jar Methenamine silver Notes #2 & #6	315W - #2 Note #3	TR 60°C Note #5	4 min. - 30 sec. - #9 Note #4
9	Wash in tap water then 3-4 changes DI	BENCH STEP
10	50ml Coplin Jar 1% gold chloride Notes #2 & #6	165W - #1 Note #3	TR 40ºC Note #5	40 sec. - #2 Note #4
11	Rinse in DI water	BENCH STEP
12	50ml Coplin Jar 2% sodium thiosulfate Notes #2 & #6	165W - #1 Note #3	TR 40ºC Note #5	40 sec. - #2 Note #4
13	Rinse in Tap then DI water	BENCH STEP
14	50ml Coplin Jar Light green solution Notes #2 & #6	165W - #1 Note #3	TR 40ºC Note #5	40 sec. - #2 Note #4
15	Dehydrate through ETOH's, clear and mount	BENCH STEP
NOTES Step #8 should reach 60ºC in the first 2 min. of the time period. 1. Adjust wattage to smooth heating curve. 2. Repeat step for 2 min. or less to improve silver development. Keep the TR at 60ºC.
NOTES Steps #10, 12 and 14 do not need to reach 40ºC in the time period.

*Or Brazilliant, Anatech

¹Requires a microwave with continuous variable wattage and a ColdSpot™ - Recommended models are a PELCO BioWave^®

REFERENCES:
Sheehan and Hrapchak, Theory and Practice of Histotechnology, 2nd ed.1980, Mosby pg. 245
Preece, A Manual for Histologic Technicians, 3rd ed., 1972, Little, Brown & Co. page 335, with modifications.

RESULTS:
Fungi and Pneumocystis - dark brown to black
Mucin - taupe to dark gray
Background - light green

Iron-Prussian Blue Stain for Ferric Iron - Microwave Procedure¹

Notes:

Place temperature probe in ColdSpot™ port when Temperature Restriction
(TR) is "None" and set TR to >50ºC
Place sample container on ColdSpot™
Check wattage setting
LED above wattage setting indicator will be RED when the magnetron is on
When a TR is set the LED will be RED until the TR is reached then it will turn GREEN (magnetron off) and cycle between RED and GREEN for the remaining time
Place temperature probe in sample container and set TR

Iron-Prussian Blue Stain for Ferric Iron - Microwave Procedure

Step	Container Reagent	Wattage Setting	Temperature Restriction	Time/Pad#
1	50ml Coplin Jar xylene Note #2	165W - #1 Note #3	None Note #1	4 min. - #8 Note #4
2	50ml Coplin Jar 95-100% ETOH Note #2	165W - #1 Note #3	None Note #1	1 min. - #3 Note #4
3	Wash in tap water to clear	BENCH STEP
4	Wash in DI water 3 changes	BENCH STEP
5	50ml Coplin Jar Ferrocyanide/HCl Notes #2 & #6	315W - #2 Note #3	TR 45°C Note #5	2 min. - #6 Note #4
6	Wash in DI water 3 changes	BENCH STEP
7	50ml Coplin Jar Nuclear Fast Red* Notes #2 & #6	315W - #2 Note #3	TR 45°C Note #5	1 min. 30 sec. - #5 Note #4
8	Wash in DI water 3 changes	BENCH STEP
9	Dehydrate through ETOH's, clear and mount	BENCH STEP
NOTES Step #5 and #7 do not require reaching 45ºC

*Or Brazilliant, Anatech

¹Requires a microwave with continuous variable wattage and a ColdSpot™ - Recommended models are a PELCO BioWave^®

REFERENCES:
Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd ed., Lee Luna (editor), 1968
Carson, F.L., Histotechnology: A Self-Instructional Text, ASCP Press, 1990

RESULTS:
Hemosiderin (iron) - blue
Nuclei and hemofuchsin - red
Background - pink

Iron-Prussian Blue Stain for Ferric Iron - Microwave Procedure¹

Notes:

Place temperature probe in ColdSpot™ port when Temperature Restriction
(TR) is "None" and set TR to >50ºC
Place sample container on ColdSpot™
Check wattage setting
LED above wattage setting indicator will be RED when the magnetron is on
When a TR is set the LED will be RED until the TR is reached then it will turn GREEN (magnetron off) and cycle between RED and GREEN for the remaining time
Place temperature probe in sample container and set TR

Step	Container Reagent	Wattage Setting	Temperature Restriction	Time/Pad#
1	50ml Coplin Jar xylene Note #2	165W - #1 Note #3	None Note #1	4 min. - #8 Note #4
2	50ml Coplin Jar 95-100% ETOH Note #2	165W - #1 Note #3	None Note #1	1 min. - #3 Note #4
3	Wash in tap water to clear	BENCH STEP
4	50ml Coplin Jar 0.5% Periodic Acid Notes #2 & #6	315W - #2 Note #3	TR 60°C Note #5	2 min. 30 sec. - #7 Note #4
5	Wash in DI water 3 changes	BENCH STEP
6	50ml Coplin Jar Schiff Reagent* Notes #2 & #6	165W - #1 Note #3	TR 45°C Note #5	2 min. - #6 Note #4
7	50ml Coplin Jar Warm Tap Water Notes #2 & #6	165W - #1 Note #3	TR 45°C Note #5	4 min. - #8 Note #4
8	Wash in tap water	BENCH STEP
9	50ml Coplin Jar Gill #1 Hematoxylin Notes #2 & #6	165W - #1 Note #3	TR 45°C Note #5	40sec. - #2 Note #4
10	Wash in DI water	BENCH STEP
11	Blue in Scott's water	BENCH STEP		1 min.
12	Wash in tap water	BENCH STEP
13	Dehydrate through ETOH's, clear and mount
NOTES Step #4 should approach 60ºC Steps #6 to #8 do not require reaching 45ºC

*Use Fresh Reagent – Destain with 0.55% Potassium Metabisulfite if needed after step #6

¹Requires a microwave with continuous variable wattage and a ColdSpot™ - Recommended models are a PELCO BioWave^®

REFERENCES:
Carson, F.L., Histotechnology: A Self-Instructional Text, ASCP Press, 1990, pg 119
Sheehan and Hrapchak, Theory and Practice of Histotechnology, 2nd ed.1980, Mosby pg.164
"A Simple Method to Restore Exhausted Schiff Reagent", The Journal of Histotechnology, Vol. 20, No. 1, March 1997, pg. 79

RESULTS:
Glycogen, neutral mucosubstances, certain epithelial sulfomucin and sialomucins, colloid material of the thyroid and pars intermedia of the pituitary, basement membranes, and fungal walls - magenta or rose to red
Nuclei - blue

Collagen & Smooth Muscle – Masson's Trichrome Stain - Microwave Procedure¹

Notes:

Place temperature probe in ColdSpot™ port when Temperature Restriction
(TR) is "None" and set TR to >50ºC
Place sample container on ColdSpot™
Check wattage setting
LED above wattage setting indicator will be RED when the magnetron is on
When a TR is set the LED will be RED until the TR is reached then it will turn GREEN (magnetron off) and cycle between RED and GREEN for the remaining time
Place temperature probe in sample container and set TR

Collagen & Smooth Muscle – Masson's Trichrome Stain - Microwave Procedure

Step	Container Reagent	Wattage Setting	Temperature Restriction	Time/Pad#
1	50ml Coplin Jar xylene Note #2	165W - #1 Note #3	None Note #1	4 min. - #8 Note #4
2	50ml Coplin Jar 95-100% ETOH Note #2	165W - #1 Note #3	None Note #1	1 min. - #3 Note #4
3	Wash in tap water to clear	BENCH STEP
4	50ml Coplin Jar Bouin's Solution Notes #2 & #6	315W - #2 Note #3	TR 60°C Note #5	6 min. - #0 Note #4
5	Wash in tap water to clear yellow	BENCH STEP
6	50ml Coplin Jar Gill #2 Hematoxylin Notes #2 & #6	315W - #2 Note #3	TR 40°C Note #5	1 min. 20 sec. - #4 Note #4
7	50ml Coplin Jar Wash in tap water to blue	BENCH STEP
8	50ml Coplin Jar Biebrich Scarlet – Acid Fuchsin Notes #2 & #6	315W - #2 Note #3	TR 40°C Note #5	40sec. - #2 Note #4
9	Rinse in DI water 3 changes	BENCH STEP
10	50ml Coplin Jar Phosphotungstic – Phosphomolybdic Sol Notes #2 & #6	315W - #2 Note #3	TR 40°C Note #5	1 min. - #3 Note #4
11	50ml Coplin Jar Analine Blue Solution Notes #2 & #6	165W - #2 Note #3	TR 40°C Note #5	40sec. - #2 Note #4
12	Rinse in tap water then DI water	BENCH STEP
13	50ml Coplin Jar 1% Acetic Acid Sol.	BENCH STEP		30 sec.
14	Dehydrate through ETOH's, clear and mount	BENCH STEP
NOTES Step #4 should reach 60ºC in 3 to 4 min. Step #6, 8, 10 and 11 do not require reaching 40ºC

*Use Fresh Reagent – Destain with 0.55% Potassium Metabisulfite if needed after step #6

¹Requires a microwave with continuous variable wattage and a ColdSpot™ - Recommended models are a PELCO BioWave^®

REFERENCES:
Sheehan and Hrapchak, Theory and Practice of Histotechnology, 2nd ed.1980, Mosby
F.L. Carson, Histotechnology: A Self-Instructional Text, ASCP Press, 1990

RESULTS:
nuclei - blue-black
collagen - blue
cytoplasm, keratin, muscle fibers, intercellular fibers - red

Paraffin Tissue Processing

Special Stains

Mucin – Alcian Blue pH 2.5 - Microwave Procedure1

Fungus & Pneumocystis – Grocott's Methenamine Silver Nitrate - Microwave Procedure1

Iron-Prussian Blue Stain for Ferric Iron - Microwave Procedure1

Iron-Prussian Blue Stain for Ferric Iron - Microwave Procedure1

Collagen & Smooth Muscle – Masson's Trichrome Stain - Microwave Procedure1

Mucin – Alcian Blue pH 2.5 - Microwave Procedure¹

Fungus & Pneumocystis – Grocott's Methenamine Silver Nitrate - Microwave Procedure¹

Iron-Prussian Blue Stain for Ferric Iron - Microwave Procedure¹

Iron-Prussian Blue Stain for Ferric Iron - Microwave Procedure¹

Collagen & Smooth Muscle – Masson's Trichrome Stain - Microwave Procedure¹