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NEW PELCO BioWave® Pro+ Microwave Tissue Processor
go to the Paraffin Tissue Processing Application Kit

Paraffin Tissue Processing


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
  1. The times and the number of reagent changes are based on processing tissues that are 2mm or less in thickness.
  2. Thicker tissues will require longer times and perhaps added dehydration and clearing steps.
  3. Formalin fixation in the microwave is done with a different setup.
  4. The second xylene step (#6) is designed to heat the samples to the same temperature as the paraffin.
  5. Paraffin is transparent (does not heat) to microwave energy. A paraffin heating tile is used to maintain the temperature during processing.
  6. True wattage, not pulsed or percent power makes the use of xylene routine. Substitutes can be used if desired (add steps to insure clearing).

The above protocol is based on tissues that are 2mm thick or less. For tissues thicker than 2mm processing times should be increased and steps added depending on the type of tissue (plant or animal) being processed.

Tissues can be processed using either the PELCO TissueVac® System (36160, Fig. 1) or the PELCO HistoWave® Cassette System (36150, Fig. 2).

Either system is placed on the PELCO ColdSpot® for processing (Fig. 3).

PELCO TissueVac™ microwave vacuum chamber PELCO HistoWave® Cassette System

The temperature restriction [Temp (°C)] for each processing step is shown in the protocol. The temperature is allowed to increase during the last clearing step (Step #6 - xylene in the above protocol) so that the system reaches the paraffin temperature. In that way the paraffin is not likely to harden during the paraffin infiltration steps. Paraffin heating tiles are offered for both systems (36160-4 for Fig. 1 and 36150-1 for Fig. 2).

Note: The PELCO HistoWave® Cassette System may also be used in the PELCO TissueVac® Vacuum Chamber, Prod. # 36160.

 



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:

  1. Place temperature probe in the PELCO ColdSpot® port when Temperature Restriction
    (TR) is "None" and set TR to >50ºC
  2. Place sample container on the PELCO ColdSpot®
  3. Check wattage setting
  4. LED above wattage setting indicator will be RED when the magnetron is on
  5. 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
  6. 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 PELCO 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:

  1. Place temperature probe in the PELCO ColdSpot® port when Temperature Restriction (TR) is "None" and set TR to >50ºC
  2. Place sample container on the PELCO ColdSpot®
  3. Check wattage setting
  4. LED above wattage setting indicator will be RED when the magnetron is on
  5. 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
  6. 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 PELCO 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:

  1. Place temperature probe in the PELCO ColdSpot® port when Temperature Restriction
    (TR) is "None" and set TR to >50ºC
  2. Place sample container on the PELCO ColdSpot®
  3. Check wattage setting
  4. LED above wattage setting indicator will be RED when the magnetron is on
  5. 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
  6. 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 PELCO 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:

  1. Place temperature probe in the PELCO ColdSpot® port when Temperature Restriction
    (TR) is "None" and set TR to >50ºC
  2. Place sample container on the PELCO ColdSpot®
  3. Check wattage setting
  4. LED above wattage setting indicator will be RED when the magnetron is on
  5. 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
  6. 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 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 PELCO 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:

  1. Place temperature probe in the PELCO ColdSpot® port when Temperature Restriction
    (TR) is "None" and set TR to >50ºC
  2. Place sample container on the PELCO ColdSpot®
  3. Check wattage setting
  4. LED above wattage setting indicator will be RED when the magnetron is on
  5. 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
  6. 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 PELCO 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


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