We compared the performance of six commercial fixatives proposed to be
formalin substitutes with the performance of buffered formalin, Clark
e's ethanol-acetic acid, and ethanol, using Fat liver, small intestine
, and kidney. We investigated the rate of penetration, mode of fixatio
n, extent of protein: and structural immobilization, quality of histol
ogy and cellular structure following routine dehydration and paraffin
embedding, and performance as a fixative for immunohistochemistry. Fur
thermore, we evaluated the effects of the various fixatives on ultrast
ructure. Only buffered formalin performed equally well on all tissues
tested. While several of the commercial fixatives appeared to preserve
liver tissue at 200 x, the preservation of kidney, intestinal villi,
and smooth muscle was unacceptable. Histological distortion, cell shri
nkage and vacuolization were prominent when the substitutes or ethanol
were used. In contrast, these artifacts were found occasionally and t
o a minor degree when buffered formalin or Clarke's fixative were used
. Immunohistochemistry demonstrated a total loss of low molecular weig
ht antigen (serotonin) and patchy reactions for high molecular weight
antigens for all fixatives except buffered formalin. The best immunost
aining was obtained by combining formalin fixation with antigen retrie
val. We conclude that none of the proposed commercial substitutes for
buffered formalin are adequate for critical histology or histopatholog
y.