REFERENCES

Crark, G.: Problems in aerial application: A com-
parison of the acute effects of endrin and carbon
tetrachloride on the livers of rats and of the residual
effects of one month after poisoning. OAM Report
No. 66-34, 1966.

Crank, G.: Problems in aerial application: A com-
parison of the effects of dieldrin poisoning in cold-
adapted and room-temperature mammals. OAM
Report No. AM 66-5, 1966.

Beruse, G. R., Powers, M. M. and Crarx, G.:
Tron hematoxylin chelates. II. Histochemistry of
myelin sheath stains. Stain Techn., 40: 235-238,
1965.

CasseLMay, W. G. B.: Histochemical Technique,
John Wiley and Sons, Inc, New York, New York,
1959.

Brruse, G. R., Powers, M. M. and Crark, G.: Iron
hematoxylin chelates. I. The Weil staining bath.
Stain Techn, 40 : 53-62, 1965.

Kornic, H., Groat, R. A. and Wrnorz, W. Fv: A
physiological approach to perfusion-fixation of tis-
sues with formalin. Stain Techn., 20: 18-22, 1945.

Baxer, J, R.: The histochemical recognition of
lipine. Quart. J. Mior. Sci., 87: 441-470, 1946,

Koenig, H.: The proteolipid nature of the neuro-
keratin network of myelin. J. Neurochem. 4: 93-
100, 1959,

BurMer, D.: Observations on histological methods
involving the use of phosphotungstic and phospho-
molybdic acids, with special reference to staining
with phosphotungstic acid/hematoxylin, Quart, J.
Mier. Sci., 103: 311-823, 1962.

67-4564



in Table 1. Unexplicably with methyl alcohol,
acetone and chloroform-methanol extraction, the
cell membranes stained deeply and after acetone
extraction red blood cells were intensely stained.

Taste 1, Extraction at —20°C for one week then 2
days in 10% formalin. 10% formalin froze at
—20°C so was kept at +8°C. 0, no stain, +,
weak stain, ++, light stain, +++, normal stain,
+++-+, overstained.

Cell Membranes Cytoplasm RBC

Chloroform-Methanol = -++-++ + lysed
(2-41)

Pyridine 0 0 lysed

Acetone Fett + ttt

Chloroform 0 + lysed

Methyl aleohol +444 + ++

10% Formalin ttt + +4+

IV. Discussion.

As shown in Table 1 the staining intensity of
the cytoplasm of the liver cells was either reduced
markedly or blocked by all the lipid solvents
used. This indicates that the compound is either
a lipid or a lipid containing complex. Extraction
with pyridine was more effective than the others
and it should be remembered that Baker’ used
loss of staining after pyridine extraction as proof
that phospholipids were stained by his acid
hematein technic. Koenig compared the staining
of spinal cord following extraction with chloro-
form-methanol (2-1) with that of unextracted
spinal cord. He found that following extraction,
the neurokeratin meshwork failed to stain with
the Weil stain. He also found that extracted
spinal cord proteolipid could be stained with the
Weil stain. Proteolipids contain phosphorus
and thus by definition are classed with phospho-
lipids. It is possible that the compound in the
liver stained by the Weil stain could be further
characterized with extraction procedures. The
increase in staining of cell membranes after
chloroform-methanol, acetone or methyl alcohol
extraction is not explainable at present nor is the
heavy staining of red cells after acetone extrac-
tion.

The staining intensity was increased by methy-
lation. Since this reaction would esterify any of
the compounds with a replaceable H-+ the pos-
sible reaction of the dye as a base is precluded.
Likewise any simple amine would be alkylated
and thus would become a substituted amine.

Similarly the staining intensity was increased by
aldehyde blockage with hydroxylamine, The fact
that blockage did not occur eliminates the CHO
group. The increase is difficult to explain but it
is possible that an addition product formed
which acted as a substituted amine. The find-
ings with acetylation are complicated. The re-
sults shown in Figure 1 are the same for both
spinal cord (Berube, Powers and Clark’) and
liver. However, Bulmer® reported that time in
the acid staining bath profoundly affected the
intensity of staining with phosphotungstic acid
hematoxylin following acetylation. A series of
slides was placed in the usual acetylation mix-
ture for three hours, rapidly hydrated and stained
for 2, 4, 8,16 and 32 minutes in the full strength
Weil stain (four times as concentrated as our
routine stain). Control slides were stained in
the same solution and for the same length of time
and were differentiated identically. The optical
density of these slides was measured by the
method previously used?, Then the density of
each acetylated slide was divided by the density
of its control. The values found were: 2 min, .87;
4 min, .92; 8 min, 1.04; 16 min, 1.07; 82 min, 1.36.
It is evident that the stain was partially blocked
initially and that overstaining developed slowly.
This is consistent with Bulmer’s® concept that
hydrolysis of the acetylated compounds occurs
in the acid staining solutions. The suggestion
might be advanced that complete acetylation
blocks the stain while after partial hydrolysis
some materials may act as a substituted amine.
The results of amine blockage and deamination
are almost conclusive in themselves in implicating
an amine as the functional group to which the
dye is attached but explanation of the results of
acid extraction are more difficult. Presumably
the acid removed some compound which then
allowed an amine to react with the dye.

There are, of course, many side reactions with
blocking procedures so it is hardly advisable to
make dogmatic statements. The total body of the
results, however, makes it possible to suggest
that the compound which decreases in cold adap-
tation but increases in poisoning with halogenated
hydrocarbons is a phospholipid containing a
secondary amine. This secondary amine is the
functional group to which the Weil stain attaches
in the liver as well as in the spinal cord.




AM 66-26

PROBLEMS IN AERIAL APPLICATION:
HISTOCHEMISTRY OF WEIL STAIN ON LIVER

George Clark

Approved by Released by
J. Roserr Diuz, M.D. P. V. Smzeet, M.D.
Cuter, Crvi, AERONAUTICAL Feperau Air SuRcEON
InstrruTs

November 1966

FEDERAL AVIATION AGENCY
Office of Aviation Medicine



Qualified requestors may obtain Aviation Medical Reports from Defense Documentation
Center. The general public may purchase from Clearinghouse for Federal Scientific
and Technical Information, U.S. Dept. of Commerce, Springfield, Va. 22151



Qualified requestors may obtain Aviation Medical Reports from Defense Documentation
Center. The general public may purchase from Clearinghouse for Federal Scientific and
Technical Information, U.S. Dept. of Commerce, Springfield, Va. 22151


PROBLEMS IN AERIAL APPLICATION:

Histochemistry of Weil Stain on Liver

I. Introduction.

The staining intensity of rat liver sections pre-
pared by the Weil method for myelin sheaths is
increased in endrin* and dieldrin? poisoning and
decreased in cold adaptation?. Knowledge of the
chemical nature of the compound or compounds
involved might well be valuable in designing
treatment for poisoning with these insecticides
and possibly also in artificial cold adaptation.
A step towards the characterization of these sub-
stances is available in the technics of histochem-
istry. In the spinal cord the Weil stain is appar-
ently specific for some phospholipid containing
a substituted amine*. Presumably the same
should be true for the liver.

IL. Methods.

The technics for acetylation, methylation,
deamination and aldehyde blockage as given by
Casselman‘ were rigorously followed. For trich-
loracetic acid extraction 0.3M acid at 90°C for 15
minutes was used and for amine blockage the
ammonium salt of rhodanilic acid method of
Berube, Powers and Clark*. In addition to the
usual control an additional slide was placed in
0.5% paratoluene sulfonic acid (solvent used for
the ammonium salt. of rhodanilic acid) for the
same length of time as in the blocking reaction.
The variant of the Weil stain used was that de-
veloped by Berube, Powers and Clark’. Livers
from rats perfused with acacia saline followed by
acacia formalin fixation® were embedded in paraf-
fin and sectioned at seven microns.

Also, small blocks of liver were rapidly excised
from a rat killed by decapitation and placed in
cold chloroform-methanol (2-1), pyridine, ace-
tone, chloroform or methyl alcohol. These were
kept at -20°C for one week and then the blocks
were placed in 10% formalin for two days at
room temperature. Control blocks were placed
in 10% formalin for nine days at 38°C. These
blocks were subsequently treated like unextracted
blocks.

II. Results.

The results obtained are shown in the bar
graph in Figure 1. In each case the correspond-
ing results from similar treatment with spinal
cord are given. These are taken from the report
of Berube, Powers and Clark*. The results with
spinal cord are shown as solid bars while those
with liver are ‘shown as striped bars. It is ap-
parent that the intensity of the stain was in-
creased with methylation, aldehyde blockage,
acetylation, paratoluene sulfonic acid and trich-
loracetic acid extraction. Deamination for 30
minutes or 24 hours resulted in decreased staining
intensity as did amine blockage with the am-
monium salt of rhodanilic acid.

All the lipid solvents used markedly decreased
the staining intensity of liver cytoplasm as shown

2

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Ga eZ Z
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contol | WA Y
Z
oa Ga Z Y
Za Z
AA
ah y
,ee g
219 Oo wv Y
AA AY
gu Ze Ga Z Y
Gu Za Za Z Y
aaa A EZ
AA AZ G
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Frevre 1. Bar graph showing staining intensity after

various blocking procedures. Solid bars—spinal cord,
striped bars—liver, a—methylation, b—aldehyde block-
age, c—acetylation, d—amine blockage, e—deamination
for 30 minutes, f—deamination for 24 hours, g—para-
toluene sulfonic acid extraction and h—perchloric acid
extraction.