by Vincent R. Klump, Jr., HT (ASCP), Histology
Services, East Haven, CT
note: Mr. Klump was instrumental in the development of a standardized, consistent, reproducible protocol
for microwave histoprocessing in the original Energy Beam Sciences H2500 Microwave Processor.
Tissue criteria:Maximum tissue size for this procedure: 3mm x 3mm x 3mm. Variations of this size can also be accommodated, but total tissue volume
should not exceed 3mm cubed.
Examples: 3mm punch biopsies, core needle biopsies, core bone marrow biopsies, shave biopsies.
Tissue
should be fixed in 10% NB Formalin for a minimum of four hours.
Equipment:
- Laboratory microwave equipped with a temperature probe
accurate to +/- 1ºC
- Microwave-safe container holding a minimum of 250ml
- (optional) microwave-safe cassette rack supplied by Energy
Beam Sciences
- 100% ethanol
- 100% isopropanol
- liquid paraffin
- boiling chips (preferably, common stones not bigger than 1cm x .5cm x
.5cm)
Procedure:
- Rinse tissue for 5 minutes in running water
- dehydrate in 100% ethanol for 15 minutes at 65ºC in microwave
- clear in
100% isopropanol for 10 minutes at 74ºC in microwave
- infiltrate (3 steps) in liquid paraffin for 5 minutes at 65ºC in microwave with
boiling chips, 5 minutes at 74º in microwave with boiling chips; fresh change of liquid paraffin for 5 minutes at 82º in microwave
with boiling chips
- transfer cassettes to clean paraffin and embed as usual
Slide criteria:
Positively (+) charged or silane treated precleaned glass slides. Tissue placed centrally on slides. Slides baked for a minimum of
1 hour at 60ºC (preferably, baked overnight at 60ºC)
Examples:
Antigen retrieval may be utilized for almost all antibodies, enhancing
staining and allowing for greater antibody dilution as well as shorter incubation times. Most antibodies which require enzyme treatment
stain remarkably well utilizing antigen retrieval procedures. Remember to always run a known positive control with each antibody and
a true negative with each case.
Equipment:
- Laboratory microwave with temperature control accurate to +/- 1ºC
- Microwave-safe staining
dish
- Microwave-safe slide rack
- Distilled water
- Citric Acid
- Sodium Hydroxide
- pH meter
Procedure:
- Xylene, 2 changes, 10 minutes each
- 100% ethanol, two changes, 1 minute each
- 2% Hydrogen Peroxide Methanol quench for 10 minutes
- 100% ethanol, two changes, 1 minute each
- 95% ethanol, two changes, 1 minute each
- 70% ethanol, 1 minute
- Rinse in distilled water, 1 minute
Citrate buffer: 10mM Citrate Buffer
(2.1g Citric Acid to 1L of distilled water, adjust pH to 6.0 with approximately 10mL of 2M NaOH). Immerse sections in citrate buffer
for 10 minutes at 100ºC in microwave.
Note: Slides *must* cool to room temperature before proceeding to IHC steps. Rinse slides in
distilled water, transfer to buffer, and go on to IHC procedure. A variety of staining techniques are suited to this procedure, including
APAAP, PAP and ABC Complex/HRP.
Note: This procedure was adapted from Dako Antibody Data Sheet for KI-1
We are all very familiar with silver stains in the microwave, but these three stains are histochemical
stains which the microwave accelerates considerably.
PAS + DIASTASE Procedure:
PAS + DIASTASE done at room temperature takes two hours.
PAS + DIASTASE aided by the microwave takes 50 minutes.
Equipment:
- Laboratory microwave with temperature control accurate to +/- 1ºC
- Microwave-safe Coplin jars
- .05% Periodic Acid
- Schiff's Reagent
- Hematoxylin, Gill III
- Diastase of malt, reagent grade, .5 gram/50ml
distilled water (mix well, make fresh)
Procedure:
- Bake, deparaffinize and hydrate slides according to standard protocol
- Microwave
slides for 15 minutes at 37ºC in diastase solution
- Rinse for 10 minutes in running water
- .05% Periodic Acid for 5 minutes
- Rinse for
1 minute in running water
- Microwave slides for 1 minute at 30ºC in Schiff's solution (be sure to either mix solution or use air bubble
agitator to equalize temperature).
- Leave slides for 5 minutes in this solution.
Note: This step *must* be done under a hood.
- Wash
in tap water at room temperature until a pink color develops (at least 1 minute).
- Counterstain in Gill III Hematoxylin for 1 minute
- Blue rinse in tap water; dehydrate, clear and mount
Note: This procedure was adapted from "The Microwave Cookbook for Microscopists",
Boon and Kok, Coulomb Press Leiden, 1992
PAS Procedure:
PAS done at room temperature takes 45 minutes. PAS aided by the microwave takes
15 minutes.
Equipment:
- Laboratory microwave with temperature control accurate to +/- 1ºC
- Microwave-safe Coplin jars
- .05% Periodic
Acid
- Schiff's Reagent
- Hematoxylin, Gill III
Procedure:
- Bake, deparaffinize and hydrate slides according to standard protocol .05%
Periodic Acid for 5 minutes
- Rinse for 1 minute in running water
- Microwave slides for 1 minute at 30ºC in Schiff's solution (be sure
to either mix solution or use air bubble agitator to equalize temperature).
- Leave slides for 5 minutes in this solution.
Note: This
step *must* be done under a hood.
- Wash in tap water at room temperature until a pink color develops (at least 1 minute).
- Counterstain
in Gill III Hematoxylin for 1 minute
- Blue rinse in tap water; dehydrate, clear and mount
Note: This procedure was adapted from "The
Microwave Cookbook for Microscopists", Boon and Kok, Coulomb Press Leiden, 1992
Fe++ Procedure:
Fe++ done at room temperature takes
30 minutes. Fe++ aided by the microwave takes less than 2 minutes.
Equipment:
- Laboratory microwave with temperature control accurate
to +/- 1ºC
- Microwave-safe Coplin jars
- 2% Potassium Ferrocyanide
- 2% Hydrochloric Acid
- Nuclear Fast Red Solution
Procedure:
- Bake, deparaffinize
and hydrate slides according to standard protocol
- Prepare working solution:
- 25ml of 2% Potassium Ferrocyanide, 25ml of 2% Hydrochloric
Acid (prepare fresh before use)
- Place slides in microwave-safe Coplin jar with 50ml of working solution
- Microwave for 40 seconds at
high power (do not use temperature probe in this step, as there is a potential for reaction between metal probe and working solution)
Note: All microwaves vary in power output; adjust microwave power level to acheive final temperature of 60ºC. Rinse well in distilled
water. Counterstain in Nuclear Fast Red for 15 seconds at 60ºC using temperature probe Rinse in tap water, dehydrate, clear and mount
Note: This procedure was adapted from "The Microwave Cookbook for Microscopists", Boon and Kok, Coulomb Press Leiden, 1992