The effects of formalin fixation on the detection of apoptosis in human brain by in situ end-labelling of DNA

Summary The technique of DNAin situ end-labelling (ISEL) for the detection of apoptotic cells has recently become the method of choice. The incorporation of a labelled nucleotide to facilitate detection into the single-stranded region of DNA cleaved by endogenous nucleases has proved to be a sensitive and straightforward technique. Previous reports have applied the technique to the study of apoptotic cells in brain tissue, which is normally subjected to relatively long-term formalin fixation. In this study we have examined the effects of long-term formalin fixation on the ability to detect apoptosis using ISEL in a variety of pathologies and in a normal rat testis. In the tissues which had been treated with overnight formalin fixation, apoptotic cells were readily identified in those pathologies where it might be expected to occur. However, in tissue which had been fixed for several weeks or more, apoptotic cells were not detectable. Samples of brain lymphoma tissue and rat testis subjected to a prospective analysis with respect to fixation time showed that the ability to detect apoptotic cells tailed off at around 3–5 weeks. In order to obviate the risk of false negative results it would be desirable to use ISEL in tissues formalin fixed for less than this period.     

The changes in crosslink contents in tissues after formalin fixation

The aim of this study was to detect crosslinks of collagen and elastin in formalin-fixed tissue, to perform quantification of these crosslinks, and to investigate the effects of formalin fixation on crosslink contents in human yellow ligament and cartilage. Pyridinoline (Pyr) is a stable and nonreducible crosslink of collagen. Pentosidine (Pen) is a senescent crosslink formed between arginine and lysine in matrix proteins, including collagen. Desmosine (Des) and its isomer isodesmosine (Isodes) are crosslinks specifically found in elastin. It is useful to measure crosslink contents of collagen and elastin as a way of investigating the properties of various tissues or their pathological changes. If it is possible to evaluate crosslinks of collagen and elastin in formalin-fixed tissues, we can investigate crosslinks in a wide variety of tissues. We used HPLC to compare the concentrations of Pyr, Pen, Des, and Isodes in the formalin-fixed tissues with their concentrations in the frozen tissues. Pyr and Pen were detected in both the formalin-fixed yellow ligament and the cartilage, and their concentrations were not significantly affected by or related to the duration of formalin fixation. Des and Isodes were detected in the formalin-fixed yellow ligament but in significantly lower amounts compared to the frozen samples. We concluded that crosslinks of collagen were preserved in formalin, but crosslinks of elastin were not preserved in it. The reason for this might be that formalin did not fix elastin tissues sufficiently or it destroyed, masked, or altered elastin crosslinks.     

The effect of hypoxia on brain gamma-aminobutyric acid levels

(1) Animals were exposed to hypoxic environments either by supplying them with breathing mixtures low in oxygen or by exposing them in a decompression chamber to simulated altitude. Both methods of producing hypoxia brought about significant increases in brain GABA levels. (2) Elevated GABA levels occurred in all species tested (mouse, hamster, rat, guinea pig, and rabbit) and reached maximal concentration 60 min after the initiation of breathing the hypoxic mixtures. Extension of the exposure beyond 60 min brought about a gradual decline in GABA level from the maximal value reached. (3) A linear relation was found between the oxygen content of the gas mixture and the elevation of GABA level. For guinea pigs, at least, the critical oxygen content required to prevent elevation of GABA level was 8.1 per cent.     

The effect of in utero hypoxia on fetal heart and brain trace metals.

This study determined the effect of in utero hypoxia on fetal heart and brain pro- and antioxidant trace metals. Dunkin-Hartley guinea pigs (50–60 days gestation) were exposed to 1 h hypoxia (7% O₂/93% N₂) followed by 4 h reoxygenation in room air. Fetal hearts and brains were harvested and analyzed for copper, iron, magnesium and zinc. Fetal brain iron was significantly increased 28% after hypoxia and 35% by 1 h posthypoxia. Fetal brain magnesium demonstrated progressive decreases of 18% by 4 h posthypoxia. No significant effects of hypoxia were observed on heart trace metals. These results indicate that prooxidant metals may be increased and antioxidant metals may be decreased in posthypoxic fetal brain during a time when these tissues may be vulnerable to oxidative injury.     

Combined effects of formalin fixation and tissue processing on immunorecognition

Abstract

It is accepted that aldehyde-based fixation of cells can affect immunodetection of antigens; however, the effects of tissue processing on immunodetection have not been analyzed systematically. We investigated the effects of aldehyde-based fixation and the various cumulative steps of tissue processing on immunohistochemical detection of specific antigens. DU145 (prostate) and SKOV3 (ovarian) cancer cell lines were cultured as monolayers on microscope slides. Immunohistochemical detection of Ki67/MIB-1 and proliferating cell nuclear antigen (PCNA) was evaluated after various fixation times in 10% neutral buffered formalin and after each of the several cumulative steps of tissue processing. The effect of antigen retrieval (AR) was evaluated concomitantly as an additional variable. Our results indicate that in addition to fixation, each of the tissue processing steps has effects on immunorecognition of the epitopes recognized by these antibodies. Extensive dehydration through ethanols to absolute ethanol had only modest effects, except for the detection of Ki67/MIB-1 in SKOV-3 cells where the effect was stronger. In general, however, establishment of a hydrophobic environment by xylene resulted in the greatest decrease in immunorecognition. AR compensated for most, but not all, of the losses in staining following fixation and exposure to xylene; however, AR gave consistent results for most steps of tissue processing, which suggests that AR also should be used for staining PCNA. The cellular variations that were observed indicate that the effects of fixation and other steps of tissue processing may depend on how antigens are packaged by specific cells.     

The effect of formalin on potato cyst-nematode, Heterodera rostochiensis

Applying 5 ml of a 38% aqueous solution of formaldehyde to 1500 ml of sandy loam prevented potato cyst-nematode, Heterodera rostochiensis, from increasing on the roots of Arran Banner potatoes planted in the soil and increased the weight of tubers produced. A comparable dosage applied to plots of sandy loam and sandy clay in winter did not prevent the nematode from multiplying on Pentland Crown potatoes but increased tuber yields. Smaller amounts of formalin had less effect on tuber yields and drenching dilute formalin solutions onto the soil was no more effective than incorporating undiluted formalin into the soil by rotavation 15 cm deep.     

The effect of altered 5-hydroxytryptamine levels on beta-endorphin content in rat brain

The purpose of the present study was to examine the effect of altering the concentration of 5-hydroxytryptamine (5-HT) on beta-endorphin (beta-Ep) content in the hypothalamus, thalamus, and periaqueductal gray (PAG)-rostral pons regions of the rat brain. The selective 5-HT reuptake inhibitor, fluoxetine (10 mg/kg), significantly lowered beta-Ep content in the hypothalamus and the PAG. Parachlorophenylalanine, which inhibits 5-HT synthesis, significantly elevated beta-Ep in all brain parts studied. Intracisternal injections of the neurotoxin, 5',7'-dihydroxytryptamine, with desmethylimipramine pretreatment, significantly increased beta-Ep content in the hypothalamus and the PAG. In adrenalectomized rats, fluoxetine significantly decreased beta-Ep levels in the hypothalamus and increased the levels in the PAG. The results indicate that 5-HT may modulate the levels of brain beta-Ep.     

The effect of some brain/gut peptides on plasma cholesterol levels in the rat

The effect of substance P (SP), bombesin (BBS), somatostatin (SRIF) and neurotensin (NT) on the plasma cholesterol levels was studied in the rat. SP and BBS had no effect, but SRIF produced a significant transient hypocholesterolemia and NT a significant transient hypercholesterolemia 15 minutes after the intravenous injections. Neither SRIF nor NT influenced the rate of the 7-hydroxylation of cholesterol. The data suggest a relation of SRIF and NT to the maintenance of the plasma cholesterol concentration.     

Effect of formalin fixation on the dry mass of isolated rat liver nuclei

Summary Effect of formalin fixation on the dry mass of isolated rat liver nuclei was studied by interference microscopy. It was found that a fixation time of 2–6 hrs prevents completely the elution of water-extractable material from nuclei. Nuclei unfixed, water-extracted for 4 hrs lose about 20% of their initial dry mass. Fixation time longer than 6 hrs resulted also in a decrease of dry mass of the nuclei. Liquid scintillation counting of ¹⁴C-formaldehyde served for finding the influence of bound formaldehyde on dry mass values. As calculated, after 2 hrs fixation with formalin the bound formaldehyde constitutes 3.6% of the dry mass of isolated untreated nuclei.     

The influence of protease digestion and duration of fixation on the immunostaining of keratins. A comparison of formalin and ethanol fixation

The effect of three proteases--trypsin, pepsin, and pronase--on the immunohistochemical staining of keratins with a broad-spectrum monoclonal antibody was investigated in paraffin sections of formalin and ethanol-fixed tissues by means of the peroxidase-antiperoxidase method. Both the length of exposure to the fixative and the duration of proteolysis were varied over a wide range. Ethanol-fixed tissues showed excellent preservation of the antigenicity of keratins, and no appreciable differences in immunostaining related to the length of fixation were found. The use of proteolytic enzymes did not improve these results; on the contrary, it caused rapid tissue disintegration. Formalin-fixed epithelial tissues stained weakly or failed to stain unless they were treated with a proteolytic enzyme. The optimal length of proteolysis varied with the degree of fixation; tissues that were fixed for long periods of time in formalin required longer exposure to a proteolytic enzyme and were more resistant to digestion than were tissues that were fixed briefly. No significant advantage of one protease over another was found in this study. We conclude that a proteolytic step must precede immunostaining for keratins if the tissue is fixed in formalin, but that the digestion period must be adjusted according to the length of exposure to the fixative. The superiority of alcohol over formalin fixation for the preservation of the antigenicity of keratins is confirmed by this study.     

The effect of some brain/gut peptides on plasma cholesterol levels in the rat

The effect of substance P (SP), bombesin (BBS), somatostatin (SRIF) and neurotensin (NT) on the plasma cholesterol levels was studied in the rat. SP and BBS had no effect, but SRIF produced a significant transient hypocholesterolemia and NT a significant transient hypercholesterolemia 15 minutes after the intravenous injections. Neither SRIF nor NT influenced the rate of the 7α-hydroxylation of cholesterol. The data suggest a relation of SRIF and NT to the maintenance of the plasma cholesterol concentration.