Lectin-binding spectra in the hyperplastic human tonsil

Summary In order to determine the effect of routine fixation on the lectin affinity of tissue structures, we used unconjugated lectins and an indirect immunoalkaline-phosphatase method for frozen sections, and the peroxidase-anti-peroxidase method for paraffin-embedded, formalinfixed tissue sections. Fourteen hyperplastic human tonsils were used, and the results of the binding spectra of each lectin were compared. In general, the binding spectrum defected in the paraffin sections was part of the broader range of affinity obtained in the frozen sections. The lectin receptors on the cell surface were especially affected by formalin fixation. On the other hand, the paraffin sections, because of their superior morphology, showed a better localization of the cytoplasmic reaction product and discriminated the cell types more accurately. Thus, the two tissue preparations are rather complementary. In the tonsil peanut agglutinin (PNA) and periodic acid/Concanavalin A (PA/Con A) proved to be suitable tools for distinguishing exactly between the crypt and the surface epithelium. Ulex europaeus agglutinin I (UEA) is a reliable endothelial marker with a strong affinity to the crypt epithelium. In the frozen sections, PNA regularly stained follicular-centre cells on their cell surface. PNA, Helix pomatia agglutinin (HPA), soybean agglutinin (SBA) and Con A stained the histiocytic population, especially PNA which additionally stained an asteroid histiocyte. This cell probably corresponds to the antigen-presenting histiocyte of the T-system.Abbreviations PNA Peanut agglutinin - UEA Ulex europaeus agglutinin I - HPA Helix pomatia agglutinin - SBA Soybean agglutinin - Con A Concanavalin A - PHA Phaseolus vulgaris agglutinin - SaR swine-anti-rabbit immunoglobulins - PaP peroxidase-anti-peroxidase-complexes - HRP horseradish peroxidase - PA periodic acid - DAB diaminobenzidine - AP alkaline phosphatase - PBS phosphate buffered saline solution - pls paraffin section - fzs frozen section - s surface - c cytoplasmic     

Unrestricted modification search reveals lysine methylation as major modification induced by tissue formalin fixation and paraffin embedding

Formalin‐fixed paraffin‐embedded (FFPE) tissue is considered as an appropriate alternative to frozen/fresh tissue for proteomic analysis. Here we study formalin‐induced alternations on a proteome‐wide level. We compared LC‐MS/MS data of FFPE and frozen human kidney tissues by two methods. First, clustering analysis revealed that the biological variation is higher than the variation introduced by the two sample processing techniques and clusters formed in accordance with the biological tissue origin and not with the sample preservation method. Second, we combined open modification search and spectral counting to find modifications that are more abundant in FFPE samples compared to frozen samples. This analysis revealed lysine methylation (+14 Da) as the most frequent modification induced by FFPE preservation. We also detected a slight increase in methylene (+12 Da) and methylol (+30 Da) adducts as well as a putative modification of +58 Da, but they contribute less to the overall modification count. Subsequent SEQUEST analysis and X!Tandem searches of different datasets confirmed these trends. However, the modifications due to FFPE sample processing are a minor disturbance affecting 2–6% of all peptide‐spectrum matches and the peptides lists identified in FFPE and frozen tissues are still highly similar.     

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 use of methyl green-pyronin staining after glutaraldehyde fixation and paraffin or araldite embedding.

Methyl green-pyronin staining has been used for localization of RNA and DNA in chick and mouse embryonic tissues and in insect larval salivary glands. Glutaraldehyde or tricholoracetic acid-lanthanum acetate (TCA-LA) was used as fixative and paraffin wax or Araldite was used as embedding medium. For good results the following are specially desirable: fixation with 2.5% glutaraldehyde, dehydration in alcohols for short time, and the use of fresh staining solutions. After TCA-LA fixation the final results are much less specific. The digestion with RNAse appears essential for the detection of RNA because pyronin does not seem to be entirely specific to RNA. The results show that glutaraldehyde a common fixative for electron microscopic work, is particularly suitable fixative for light microscopic cytochemical investigations if followed by methyl green-pyronin staining; furthermore, methyl green-pyronin staining after glutaraldehyde fixation can be carried out on Araldite sections.     

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 lectin binding sites on the plasma membrane components of human lymphoblastoid cell lines.

The plasma membrane components of five human B-cell lines and three human T-cell lines were separated by dodecyl sulfate polyacrylamide gel electrophoresis, incubated with the radioactive labeled lectins from lentil, castor bean, wheat germ, Phaseolus bean, peanut, gorse and the Roman snail and the molecular weights of the binding sites determined. The lentil, castor bean and wheat germ lectin bound to multiple components from molecular weights (Mr) 20 000 to 200 000 within the plasma membranes, whereas peanut lectin bound preferentially to glycoproteins of Mr 150 000 and 83 000 in B-cells, and 150 000 and 130 000 in T-cells. The gorse lectin bound to a 220 000 component in B-cells which was not labeled in T-cells.     

DNA quantification of squamous cell carcinoma of the oesophagus by flow cytometry and cytophotometric image analysis using formalin fixed paraffin embedded tissue.

This is the first comparative study of DNA quantification of oesophageal squamous cell carcinoma by flow cytometry (FCM) and image cytometry (ICM) using formalin fixed paraffin embedded tissue. The potential advantages of ICM include the identification of a reliable control cell population; avoidance of non-tumour stromal and inflammatory cell nuclei, nuclear fragments, degenerate cell nuclei and doublets, triplets etc., which are not possible with FCM using archival tissue. Twenty-eight cases, all of the same stage (stage 2a) and similar grade (well or moderately differentiated) were analysed. The cases were separated into two groups, those that had succumbed to tumour in less than 18 months (group A) and those that were tumour free at least 18 months post-resection (group B). Using ICM all 28 tumours yielded interpretable histograms by comparison to 25 of 28 using FCM. Aneuploidy was identified in 100% of cases in group A using ICM (in comparison to 73% by FCM) and in 73% of group B using ICM (in comparison to 44% by FCM). Any tumour aneuploid by FCM was also aneuploid by ICM. Nine cases aneuploid by ICM were euploid by FCM. The mean 5C exceeding rate (% of cells whose nuclei contain a DNA mass equivalent to > 5 sets of 23 chromosomes) was 21% in group A and 14% in group B (P < 0.01). Euploidy was confined to tumours of those patients disease free for more than 18 months. The conclusions of this study are that: firstly, ICM is superior in its yield of interpretable histograms to FCM using formalin fixed paraffin embedded tissue; secondly, ICM is more sensitive in the identification of aneuploid stemlines than FCM; and thirdly, euploid tumours (as detected by ICM) appear to have a better prognosis than aneuploid tumours of similar stage and grade.     

Histologic changes in tissue components of the hyperplastic thyroid gland during its involution in the rat

Male Fischer rats were fed a low-iodine diet containing thiouracil for 21 days to produce hyperplastic thyroid glands, and then fed a high-iodine diet for various time intervals, from 5 hr to 180 days, in order to study the morphological changes that occur during involution. Thyroids were fixed by perfusion fixation and embedded in Epon. Sections were examined by light microscopy. Initially at 0 days of involution (at the time of the change to the high-iodine diet), follicular lumens were very narrow and capillary lumens were very wide. The capsule was thick and infiltrated with mononuclear leukocytes. No obvious changes occurred for 1 day after the change in diet, but shortly thereafter capillary lumens began to narrow. By 4 days, most capillary lumens were close to normal size; capillaries formed a more or less normal bed except that many were embedded in a relatively thick or wide interfollicular matrix. This matrix was largely gone by 21 days. Between 1 and 21 days, follicular lumens dilated progressively as colloid accumulated. The density of staining of the accumulated colloid varied from follicle to follicle, and this variation was also observed in older controls. Inflammatory cells gradually disappeared from the capsule and most were gone by 15 days. Starting at approximately 15 days and continuing to 180 days, one or more disintegrating cells were found in some lumen profiles. Colloid goiters were not observed in these rats even after several months of involution. Some lumens were rather large, however, and small fractions of the follicles, both small and large, were bounded by flat cells and resembled "cold" follicles morphologically.     

Differential sensitivity of the microtubule-associated protein, tau, in Alzheimer's disease tissue to formalin fixation

Immunohistochemistry of formalin-fixed human Alzheimer's disease (AD) tissue using an anti-tau antibody (Tau-1) reveals staining of neurofibrillary tangles (NFTs) and neuritic plaques (NPs), whereas normal axonal staining is less apparent. In this study, we used a combined biochemical and histochemical approach to assess effects of formalin on immunoreactivity of AD tau. Nitrocellulose blots were treated with fixative to mimic conditions used with tissue sections, a method that might be generally useful for assessing antigen sensitivity to different fixatives. A progressive decrease in Tau-1 immunoreactivity of the tau bands on a Western blot was observed with increasing times of formalin fixation. Phosphatase-digested blots demonstrated an increase in Tau-1 immunoreactivity compared to control blots. These results mimic the phosphatase-sensitive Tau-1 immunohistochemical staining of formalin-fixed AD tissue slices previously reported. Fixation of AD tissue with periodate-lysine-paraformaldehyde (PLP) preserves axonal tau antigenicity. Phosphatase digestion of PLP-fixed AD tissue enhances Tau-1 immunoreactivity of NFTs and NPs but does not alter axonal staining. These results indicate that axonal form(s) of tau are more sensitive to formalin fixation than pathology-associated tau. In addition, a modification of AD tau in pathological structures may protect it from the effects of formalin with regard to Tau-1 antigenicity.     

The accumulation of immunoglobulins in the human tonsil.

The concentration of 5 types of immunoglobulins in both serum and the faucial tonsil was determined. The level for all types was significantly higher in the tonsil. IgG was detected as the main immunoglobulin in the tonsil, while the concentration of the other types was in about the same range. With the shortening of the biological half-live the accumulation index increases. The results give evidence for the antibody formation in the tonsil.     

Effect of thyrotropin-releasing hormone on the carbohydrate structure of secreted mouse thyrotropin. Analysis by lectin affinity chromatography

Thyrotropin (TSH) is a glycoprotein hormone whose secretion from the anterior pituitary is regulated, in part, by the hypothalamic tripeptide thyrotropin-releasing hormone (TRH). We have used serial lectin affinity analysis to explore whether TRH, in addition to promoting TSH secretion, alters the carbohydrate structure of secreted TSH. Hypothyroid mouse hemipituitaries were incubated in medium containing [3H] mannose, [3H]glucosamine, or [3H]fucose either with or without 10(-7) M TRH. TSH was immunoprecipitated, proteolytically digested into glycopeptides, and chromatographed on serial lectin-Sepharose columns. Under basal conditions, 37% of secreted [3H]mannose-labeled TSH glycopeptides failed to bind to concanavalin A (ConA)-Sepharose, 55% bound and eluted with 10 mM alpha-methylglucoside, and 8% bound and eluted with 500 mM alpha-methylmannoside. Approximately 35% of glycopeptides not binding to ConA-Sepharose were bound by pea lectin-Sepharose, suggesting the presence of certain core fucosylated triantennary complex oligosaccharides. TRH caused a 2-fold increase in secretion of [3H]mannose-labeled TSH glycopeptides due almost exclusively to a specific increase in structures that bound to ConA-Sepharose and eluted with 10mM alpha-methylglucoside, corresponding to biantennary complex or unusual hybrid species. There was no change in the distribution of intrapituitary TSH glycopeptides with TRH. Acid hydrolysis of secreted proteins showed little metabolism of the tritiated sugar precursors, except for a 20% conversion of [3H]mannose to [3H]fucose. Moreover, ConA-Sepharose chromatography of secreted [3H]glucosamine- and [3H]fucose-labeled TSH glycopeptides showed similar increases in ConA-Sepharose binding with TRH as noted with [3H]mannose labeling. Subsequent lectin analysis of secreted [3H] mannose-labeled TSH glycopeptides on erythroagglutinating phytohemagglutinin-Sepharose and leukoagglutinating phytohemagglutinin-Sepharose disclosed no significant differences in TRH-treated versus control samples. These data suggest that secreted mouse TSH has greater carbohydrate heterogeneity than has been recognized previously. In addition, TRH in vitro promotes the secretion of specific TSH molecules apparently enriched in biantennary complex or unusual hybrid oligosaccharides.     

In situ RT-PCR allows the detection of ornithine decarboxylase mRNA in paraffin embedded archival human hyperplastic breast tissues

Expression of ornithine decarboxylase (ODC) is induced by c-Myc oncoprotein and is required for cell proliferation and tumour growth. We have studied the expression of ODC mRNA by in situ hybridisation and in situ RT-PCR in archival human hyperplastic breast tissues. A very low signal was detected by in situ hybridisation, while the in situ RT-PCR on human breast archival tissues demonstrated an over-expression of ODC mRNA in epithelial cells characterised by some degree of hyperplasia, maintaining the morphology of the archival tissue intact despite the multiple steps of fixation, permeabilization and thermal cycling.     

The influence of fixation and tissue preparation on the immunohistochemical demonstration of fibronectin in human tissue

The influence of fixation and tissue preparation on the immunohistochemical localization of human fibronectin in gastrointestinal tract tissue has been examined using indirect immunoperoxidase technique. The most optimal staining result with strong intensity and well defined localization was obtained on frozen sections of unfixed material. Nearly identical results with improved morphology were obtained when staining paraffin sections of tissue fixed in 96% ethanol, 96% + 1% acetic acid and absolute acetone. All other fixatives tested, 10% neutral buffered formalin. Lillie's AAF, Bouin's fixative, Clarke's fixative, 4% formaldehyde, 4% formaldehyde + 0.5% cetylpyridiniumchloride (F-CPC), 4% formaldehyde +0.1% glutaraldehyde gave unsatisfactory results. However, proteolytic digestion with pepsin of paraffin sections prior to staining of buffered formalin and F-CPCfixed material gave results comparable with those obtained on unfixed frozen sections are regards definition of the staining whereas staining intensity was decreased in some degree. No improvement was observed when using proteolytic digestion of tissue fixed in other fixatives.     

The influence of fixation and tissue preparation on the immunohistochemical demonstration of fibronectin in human tissue

Summary The influence of fixation and tissue preparation on the immunohistochemical localization of human fibronectin in gastrointestinal tract tissue has been examined using indirect immunoperoxidase technique. The most optimal staining result with strong intensity and well defined localization was obtained on frozen sections of unfixed material. Nearly identical results with improved morphology were obtained when staining paraffin sections of tissue fixed in 96% ethanol, 96%+1% acetic acid and absolute acetone. All other fixatives tested, 10% neutral buffered formalin, Lillie's AAF, Bouin's fixative, Clarke's fixative, 4% formaldehyde, 4% formaldehyde+ 0.5% cetylpyridiniumchloride (F-CPC), 4% formaldehyde +0.1% glutaraldehyde gave unsatisfactory results. However, proteolytic digestion with pepsin of paraffin sections prior to staining of buffered formalin and F-CPCfixed material gave results comparable with those obtained on unfixed frozen sections as regards definition of the staining whereas staining intensity was decreased in some degree. No improvement was observed when using proteolytic digestion of tissue fixed in other fixatives.