Effects of Microwave Fixation and
Decalcification on Rodent Tissue
April L. Marr and Anthony Wong
Pfizer Global Research and Development, San Diego, CA
Abstract
The histological use of laboratory microwaves was proposed by Mayers in 1970. Since then, microwaves have
become widely used in histology laboratories for fixation,
decalcification, and processing; however, protocols must be
carefully optimized to ensure the maintenance of optimal
cellular morphology and antigenicity. In the current study,
we sought to decrease laboratory turnaround time and optimize specimen preservation conditions by using microwave
fixation. Mouse and rat tissues were harvested and divided
into two groups (A and B). Tissues in group A were fixed
for 24 h in 10% neutral buffered formalin (NBF), whereas
tissues in group B were fixed for 2 h in 10% NBF before
being microwaved at 50°C for varying times. Tissues from
the two groups were compared by a pathologist. We found
that the optimal microwave formalin-fixation times necessary to preserve morphology were 29 min for mouse tissues
and 30 min for rat tissues. These times allowed specimens
to be placed on a processor 3 h after tissue collection and
processed for embedding the following day. We also determined optimal times for microwave decalcification of mouse
and rat sternums and femurs. Decalcification of rat femurs
with the combined use of microwave fixation and decalcification saved nearly 2 days of specimen turnaround times.
To assess the effect of microwave fixation on antigenicity,
rat intestine samples from groups A and B were stained with
Ki-67 and caspase 3 antibodies. When compared with tissues
that had been bench-fixed for 24 h, microwave-fixed tissues
showed no loss of antigenicity. We conclude that rodent fixation and decalcification times can be considerably reduced
with the aid of a microwave, thereby decreasing study turnaround times, minimizing costs, and improving efficiency.
Microwave fixation would be a valuable investment for any
laboratory seeking to save time while maintaining optimal
cellular morphology and antigenicity. (The J Histotechnol
32(4):190–192, 2009)
Introduction
Microwaves, or electromagnetic radiowaves, have been in
use for more than 60 years. Over the decades, microwaves
Address reprint requests to April Marr, Pfizer Global Research and
Development, 10646 Science Center Drive, San Diego, CA 92121. E-mail:
[email protected]
190
have been found to have many uses such as for RADAR,
food preparation and, more recently, in the histology laboratory. The histological use of laboratory microwaves was proposed by Mayers in 1970 (1) for the process of tissue fixation.
The standard home and laboratory microwave are most often
used at a frequency of 2.45 GHz. According to Giberson and
Galvez (2), the household microwave differs from the laboratory microwave in the duty cycle time of the magnetron
(the time the magnetron is on divided by the time base).
Laboratory microwaves generally have a cycle time of 1 s,
whereas a conventional microwave has a much longer cycle
of approximately 10 s or more. If a laboratory microwave
operates at a 50% duty cycle, the magnetron is on for half a
second and then off for half a second. A household microwave at the same percentage duty cycle is on for 5 s and off
for 5 s. The magnetron of the household microwave is off for
a much longer period of time, which leads to a greater fluctuation in temperature. For this reason, the laboratory microwave is much more suitable for reproducible results during
scientific research than the household microwave. The main
focus of our study was to determine optimal laboratory
microwave decalcification and formalin-fixation times for
rodent tissues by using routinely used reagents and to investigate whether those tissues were adversely affected by the
microwave exposure by comparing the results to routine processes. We also sought to determine whether tissues fixed in
the microwave provided comparable results with those of
routine processes when performing immunohistochemistry
(IHC).
Materials and Methods
Microwave Fixation Protocol
Various mouse and rat tissues were collected, including
the major organs. The tissues collected from both species
were adrenal gland, brain, heart, gastrointestinal tract, kidney, liver, lung, mesentery, skeletal muscle, pancreas, salivary gland, skin, spleen, and thymus. The tissues were
divided into two groups (A and B) per species. All tissues
were trimmed to a thickness of approximately 3 mm.
Group A was fixed for 24 h in 10% neutral buffered formalin (NBF) at room temperature. Group B was fixed for
2 h after necropsy in 10% NBF, and then all of the tissues
were subdivided into their respective microwave fixation time
intervals, i.e., 25–35 min at 1-min intervals, and microwaved
Microwave Fixation, Decalcification on Rodent Tissue / Marr and Wong
at 50°C in 10% NBF with the use of the Milestone RHS 1
Microwave Histoprocessor (Figure 1).
Tissues (group B) were taken out of the microwave at
their respective times and processed routinely overnight,
together with group A tissues before paraffin embedding
and sectioning (Figure 2). Sections for pathology review
(Figure 3) were cut at a thickness of 4 µm from all tissue
blocks and stained with hematoxylin & eosin (H&E) with
the use of the Leica XL Autostainer (Leica Microsystems
Inc., Bannockburn, IL).
Microwave Decalcification Protocol
Sternums and femurs were collected from both mice and
rats and fixed with the use of 10% NBF at room temperature
for 24 h. Rat sternums and mouse femurs were placed in
Cal-Rite decalcification fluid (Richard-Allan Scientific,
Kalamazoo, MI), microwaved at 37°C, and manually checked
for complete decalcification at 30-min intervals. Rat femurs
were placed in Cal-Rite decalcification fluid, microwaved at
37°C, and manually checked for complete decalcification at
1-h intervals. After decalcification, the bone specimens were
Figure 1. Milestone RHS 1 Microwave Histoprocessor.
Figure 2. Total microwave times include an 8-min ramp-up time to reach
desired temperature.
The Journal of Histotechnology / Vol. 32, No. 4 / December 2009
Figure 3. H&E stain. (A) 24-h fixed rat kidney, (B) 30-min microwave
fixed rat kidney, (C) 8-h routinely decalcified rat sternum, and (D) 4-h
microwave decalcified rat sternum. All figures 400× total magnification.
processed routinely overnight, paraffin-embedded, and sectioned for standard H&E analysis by pathologists for cell
integrity. After microscopic analysis, any bone specimens
that were determined not to be completely decalcified were
subjected to a repeat of the aforementioned protocols with
the use of longer time intervals to ensure that full decalcification was achieved.
Immunohistochemistry and Microwave Fixation
A large section of duodenum and jejunum was taken from
a rat and placed immediately in 10% NBF at room temperature. After 2 h of fixation, the tissues were subdivided and
placed into cassettes (groups C and D). Group C cassettes
were allowed to fix for a total of 24 h at room temperature.
Group D cassettes were microwaved in 10% NBF at 50°C for
the predetermined time. Tissues from both groups were processed routinely overnight, paraffin-embedded, and sectioned
at 4 µm. To ensure adequate drying and prepare for IHC
staining, the sections were placed in a 40°C oven overnight.
Sections from groups C and D were stained by the use of
a Leica BondTM IHC automated instrument with Ki-67 antibody for cell proliferation (NeoMarkers Inc., Fremont, CA)
and an antibody against cleaved caspase 3 for apoptotic cells
(Cell Signaling Technology, Danvers, MA). Antigen retrieval
in tissues was done with Bond Epitope Retrieval Solution 2
for 20 min, followed by endogenous protein blocking with
Protein Block, Serum-Free for 10 min (Dako, Carpinteria,
CA). The optimal working dilutions of Ki-67 and caspase 3
were 1:800 and 1:200, respectively. Both were incubated for
40 min. The primary antibodies were detected with the use
of Bond Polymer Refine Detection System for the instrument default time. A known tissue positive control tissue
was used for each primary antibody, and a negative control
was generated by substituting each primary antibody with a
rabbit serum IgG.
Results
Fixation Results
A direct comparison between 24 h-fixed (group A) and
microwave-fixed tissues (group B) of cellular morphology
and artifacts was performed (Table 1). After review by several pathologists, the optimal microwave fixation times with
the least artifacts for mouse and rat tissues were determined
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Table 1. Comparison of fixation and decalcification times for routine and microwave tissues
Rat
Mouse
Standard fixation, 10% NBF
24 h
24 h
Routine decalcification of sternum
8h
1 h and 30 min
Routine decalcification of femur
25 h, trim, additional 17 h
18 h
74 h total for bench procedures
43 h and 30 min total for bench
procedures
10% NBF microwave fixation at 50°C
30 min
29 min
Microwave decalcification of sternum at 37°C
4h
30 min
Microwave decalcification of femur at 37°C
27 h
3 h and 30 min
31 h and 30 min total for
4 h and 29 min total for microwave
microwave procedures
procedures
Time saved per species
42 h and 30 min
39 h and 1 min
to be 29 min and 30 min, respectively. Tissues (e.g., eye, testis, epididymis) that are not usually retained in 10% NBF
were not processed with microwave fixation.
Decalcification Results
After many trials, the optimal decalcification times were
determined to be as follows: rat sternum, 4 h; rat femur, 27 h;
mouse sternum, 30 min; mouse femur, 3 h and 30 min.
Immunohistochemistry Results
The IHC labeling of both Ki-67 and caspase 3 in routine
and microwave-fixed tissues was comparable. A slight reduction of background can be seen in the microwave section
stained with Ki-67 (Figure 4B, arrow).
Conclusion
After analyzing the fixation, decalcification, and
IHC results, we conclude that the use of the Microwave
Histoprocessor does not adversely affect cell morphology
or the detection of Ki-67 and caspase 3 proteins under the
optimal conditions determined in this study. The quality
of microwave-fixed tissues, at the respective optimal time
points, is comparable with routinely fixed tissues. With
the ability to have entirely fixed tissues 3 h after necropsy,
tissues can be harvested in the morning, placed on the
processor in the afternoon, and embedded the following
morning.
Review by a pathologist indicated that microwaving bone
specimens at 37°C in Cal-Rite decalcifying fluid produced
samples that are comparable with those processed by routine
decalcification procedures. Rodent bone decalcification
times can be considerably reduced with the aid of a microwave, saving days from turnaround times.
The IHC results proved that, under specific conditions,
microwave fixation does not adversely affect the immunohistochemical detection of Ki-67 and caspase 3 proteins.
Microwave fixation provides the same staining results as
routinely fixed tissues and, in some cases, better staining
results. In the detection of Ki-67 nuclear antigen, background staining is reduced and provides a more desirable
result with microwave fixation. Laboratory microwaves are
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Figure 4. (A) Ki-67, 24-h fixed rat duodenum; (B) Ki-67, 30-min microwave-fixed rat duodenum, (C) caspase 3- to 24-h fixed rat jejunum, (D) caspase 3- to 30-min microwave-fixed rat jejunum. All figures 400× total
magnification.
essential tools for any histology laboratory seeking to save
time, minimize costs, and improve efficiency.
Acknowledgments
The authors thank the members of the La Jolla DSRD histology group for their encouragement and technical support
throughout this project. They would also like to thank James
Watson (Genomics Institute of the Novartis Research
Foundation, San Diego) for sharing his microwave knowledge and expertise, and especially Winston Evering and his
team (DSRD, Pfizer) for his willingness to provide pathology analysis and feedback.
References
1. Mayers CP: Histological fixation by microwave heating. J Clin
Pathol 23:273–275, 1970.
2. Giberson R, Galvez J: Controlled microwave radiation is a versatile technology. Presented at the California Society for
Histotechnology Annual Symposium Convention. Millbrae,
California. May 14, 2009.
Microwave Fixation, Decalcification on Rodent Tissue / Marr and Wong