Combined Alizarin Red-Reticulum Stain for Tissue
Localization of Calcium Deposits
AHMAD ELBADAWI, M.D., LINDA A. MUSTO, HT(ASCP), AND OTTO MICHAEL LILIEN, M.D.
Elbadawi, Ahmad, Miistd, Linda A., and Lilien, Otto Michael:
Combined alizarin red-reticulum stain for tissue localization
of calcium deposits. Am J Clin Pathol 75: 355-356, 1981.
A method combining alizarin red and a modified silver stain
for reticulum fibers in tissue sections is described. The method
allows precise localization of calcium deposits and crystals
in relation to epithelial and interstitial connective tissue elements. The deposits and their spatial relationships are clearly
discernible by light microscopy, especially with polarization.
The method is particularly useful for investigating calculogenesis and may prove to be of value in the study of the mechanism
and evolution of pathologic calcification in various organs.
(Key words: Calculogenesis; Tissue calcium deposits; Alizarin
red; Calcium-reticulum stain.)
Departments of Pathology and Urological Surgery,
Upstate Medical Center, Syracuse, New York
(2) Alizarin red. Stir 1 g of alizarin red St into 100 ml
distilled water until only a few small grains of the dye
remain undissolved. Add approximately 12 ml of 1%
ammonium hydroxide (specific gravity, 0.896) slowly
with constant stirring, to obtain a pH of 6.36-6.40.
The final solution is stable for one month.
The following solutions must be prepared just before use:
(1) Acidified potassium permanganate. Dissolve
0.25 g of potassium permanganate in 100 ml of distilled water; add three drops of concentrated sulfuric acid.
(2) Ammoniacal silver solution. To 10 ml of 10%
aqueous silver nitrate add 2.5 ml of 10% aqueous potassium hydroxide; add concentrated ammonium hydroxide (specific gravity, 0.896) drop by drop (30-50
drops or 1 -1.7 ml) with constant shaking, until the precipitate just dissolves, and then bring the volume to 45
ml with distilled water.
IN A STUDY of the early stages of renal calculogenesis
in an experimental rat model, we sought a staining
procedure that allows easy and clear-cut differentiation
of renal tubules from interstitial tissue elements, and
at the same time clearly demonstrates calcium deposits
and crystals. After repeated trials, a technic combining
alizarin red1 and a modified silver stain for reticulum
fibers2 has been standardized. The. technic yields excellent results consistently, and is applicable to organs
other than the kidney in both experimental and human
material.
Staining
Materials and Methods
Sections (4-6 /urn) of rat kidney fixed in formalin
or Bouin's solution and processed routinely by the
Autotechnicon® were used. The following aqueous
solutions can be kept in stock: (1) 2% potassium
metabisulfite; (2) 20% formalin (8% formaldehyde);
(3) 0.2% gold chloride; (4) 5% sodium thiosulfate. The
following dye solutions also can be kept in stock:
(1) Nuclear fast red. Dissolve 0.1 g of nuclear fast
red* in 100 ml of 5% aqueous aluminum sulfate with
the aid of heat; cool, filter, and add a few grains of
thymol.
Received May 28, 1980: received revised manuscript and accepted
for publication July 8, 1980.
Address reprint requests to Dr. Elbadawi: Department of
Pathology, State University of New York, Upstate Medical Center,
766 Irving Avenue, Syracuse; New York 13210.
* Nuclear fast red (Kernechtrot), Chroma brand, was obtained
from Roboz Surgical Instruments Co., 810 18th St. N.W.,
Washington D.C.
Procedure
(1) Deparaffinize and hydrate to distilled water; remove picric acid completely in Bouin-fixed tissues.
(2) Oxidize with acidified potassium permanganate
for 2 min; rinse in three changes of distilled water.
(3) Reduce in 2% potassium metabisulfite for 1 min;
wash well in running tap water; rinse in four changes
of distilled water.
(4) Transfer to ammoniacal silver solution for 2 min;
rinse in three changes of distilled water.
(5) Reduce in 20% formalin for 3 min; wash in running tap water for 3 min; rinse in distilled water.
(6) Tone in 0.2% gold chloride for 2 min; rinse in
distilled water.
(7) Fix in 5% sodium thiosulfate for 1 min; wash
well with tap water; rinse in distilled water.
(8) Place in 95% ethanol; drain off excess.
t Alizarin red was obtained from Allied Chemicals National
Biological Stains and Reagents Department, Morristown, New
Jersey.
0002-9173/81/0300/0355 $00.60 © American Society of Clinical Pathologists
355
356
ELBADAWI, MUSTO, AND LILIEN
A.J.C.P. • March 1981
FIG. 1. Renal tubules with interstitial and intraepithelial calcium deposits (arrows): peritubular and interstitial reticulumfibersare clearly
demonstrated. Photography under polarization. x240.
(9) Stain in alizarin red solution for 10 min; remove excess stain by rinsing in six changes of distilled
water.
(10) Counterstain with nuclear fast red for V/i min;
rinse in distilled water.
(11) Dehydrate in two changes each of 95% and absolute ethanol; clear in three changes of xylene; mount
in Permount®.
Results
Calcium deposits appear orange by light microscopy
and polarize in various shades of white to pink or
orange. Both nuclei and cytoplasm are stained light
red, the former remaining easily discernible. Reticulum
fibers stand out by their intense black color, even with
polarization (Fig. 1).
Comments
Although this procedure yields excellent results,
some sections may fall off the slides if the concentration of sulfuric acid in the acified potassium permanganate solution or that of ammonium hydroxide
in the ammoniacal silver solution is higher than indicated in the procedure. Substituting acetic for sulfuric acid, or using a combination of both acids in
the potassium permanganate solution, averted detachment of the sections but led to increased background
silver staining with a tendency to form finely granular
precipitates. Separation of the sections is easily
avoided by careful preparation of both solutions, and
by the addition of 5% gelatin solution (approximately
35 ml) to the warm water bath in which the sections
are spread before mounting on slides. Although the
color of nuclei is close to that of the cytoplasm, trials
with stains other than nuclear fast red, including
hematoxylin, celestine blue, methyl green, and various
azures, alone or in combination with cytoplasmic dyes,
gave inferior results.
References
Luna LG: Manual of histologic staining methods of the Armed
Forces Institute of Pathology. Third edition. New York,
McGraw-Hill, 1968, pp 175-176
Churukian C: Modified Gomori"s method for staining reticulum
and collagen. Histo-Logic 2:23, 1972