Effects of tissue processing techniques in acoustical (1.2 GHz) and light microscopy

Summary In this study the influence of various tissue processing and staining techniques on the acoustical properties of liver tissue was investigated. A qualitative study was performed using ultrasound attenuation as the imaged parameter of a combined optical/acoustical microscope with a 1.2 GHz transducer. Images were made of three sets of adjacent liver sections (6 m in thickness) which were prepared in ten different ways: fixed by alcohol or formalin; stained by hematoxylineosin (HE), toluidine blue (TB) or non-stained; sectioned by a cryostat or by a paraffin microtome. It was concluded that the images obtained from cryostat sections were of much higher quality than those from paraffin sections. Images obtained from sections that were sectioned while embedded in paraffin displayed no detail at all. No consistent effect was noticed with respect to staining by HE or TB. Alcohol fixed sections gave more detailed images than formalin fixed sections. Formalin fixation in combination with cryostat sectioning yielded many cytoplasmic vacuoles.     

Automatic quantification of immunohistochemically stained cell nuclei based on standard reference cells.

A fully automatic method for quantification of images of immunohistochemically stained cell nuclei by computing area proportions, is presented. Agarose embedded cultured fibroblasts were fixed, paraffin embedded and sectioned at 4 microm. They were then stained together with 4 microm sections of the test specimen obtained from bladder cancer material. A colour based classifier is automatically computed from the control cells. The method was tested on formalin fixed paraffin embedded tissue section material, stained with monoclonal antibodies against the Ki67 antigen and cyclin A protein. Ki67 staining results in a detailed nuclear texture with pronounced nucleoli and cyclin A staining is obtained in a more homogeneously distributed pattern. However, different staining patterns did not seem to influence labelling index quantification, and the sensitivity to variations in light conditions and choice of areas within the control population was low. Thus, the technique represents a robust and reproducible quantification method. In tests measuring proportions of stained area an average standard deviation of about 1.5% for the same field was achieved when classified with classifiers created from different control samples.     

Effects of prolonged water washing of tissue samples fixed in formalin on histological staining

The effects of prolonged water washing after fixation for 48 h in 10% (v/v) phosphate-buffered neutral formalin on the quality of representative histological staining methods were evaluated using samples of liver, kidney, spleen and thymus collected from three male Crl:CD(SD)(IGS) rats and one male beagle dog. Because door-to-door courier services in Japan prohibit handling formalin, our goal was to confirm that formalin fixed wet tissue samples could be stored in tap water rather than formalin during transportation of the samples without decreasing the quality of their staining or immunohistochemistry. Each tissue sample was allocated randomly to one of three groups: 12 min, 3 days and 7 days of washing in running tap water; samples then were routinely embedded in paraffin and sectioned. The sections were stained with hematoxylin and eosin, periodic acid-Schiff, azan, and the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. Immunohistochemical staining for Factor VIII, ED-1 and CD3 also was assessed. Prolonged water washing for up to 7 days did not affect the morphology or stainability by standard histological methods, or the intensity and frequency of positive reactions using the TUNEL method. Only immunohistochemical staining of Factor VIII was altered in both the rat and dog sections after 7 days of water washing. The intensity of positive reactions of Factor VIII immunohistochemistry after 7 days water washing was still strong enough to detect microscopically. Therefore, prolonged water washing for up to 7 days after formalin fixation does not have seriously detrimental effects on the quality and characteristics of paraffin sections stained by various methods, including immunohistochemistry.     

Application of Microwave Irradiation to Immunohistochemistry: Preservation of Antigens of the Extracellular Matrix

Microwave irradiation as a means of fixation was evaluated for the preservation of extracellular matrix antigens such as collagen III, IV, fibronectin and laminin in both lung and liver specimens. Small tissue samples were placed in normal saline or periodatelysine-paraformaldehyde (PLP) and irradiated for 30 sec to bring them to a temperature of 50 C. The tissue was then processed rapidly in a tissue processor adjusted to a 2 hr cycle and embedded in paraffin. Sections were immunostained. For comparison, routine cryostat sections as well as sections of formalin fixed tissue were used. Microwave irradiation in saline gave excellent morphological detail, comparable to that in formalin fixed tissue. All four antigens evaluated were well preserved without the necessity of prior pepsin digestion. Microwave fixation is promising for preservation of antigenicity and morphological detail, and considerably reduces the time required for processing.     

Application of microwave irradiation to immunohistochemistry: preservation of antigens of the extracellular matrix

Microwave irradiation as a means of fixation was evaluated for the preservation of extracellular matrix antigens such as collagen III, IV, fibronectin and laminin in both lung and liver specimens. Small tissue samples were placed in normal saline or periodate-lysine-paraformaldehyde (PLP) and irradiated for 30 sec to bring them to a temperature of 50 C. The tissue was then processed rapidly in a tissue processor adjusted to a 2 hr cycle and embedded in paraffin. Sections were immunostained. For comparison, routine cryostat sections as well as sections of formalin fixed tissue were used. Microwave irradiation in saline gave excellent morphological detail, comparable to that in formalin fixed tissue. All four antigens evaluated were well preserved without the necessity of prior pepsin digestion. Microwave fixation is promising for preservation of antigenicity and morphological detail, and considerably reduces the time required for processing.     

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.     

Tape transfer sectioning of tissue microarrays introduces nonspecific immunohistochemical staining artifacts

Abstract

Tissue microarrays place tens to hundreds of formalin fixed, paraffin embedded tissue cores into a paraffin block in a systematic grid pattern that permits their simultaneous evaluation in a single section. The fragmented nature of the tissue cores often makes sectioning of tissue microarrays difficult so that the resulting disks of tissue lose their shape, fracture or fall out of the paraffin section altogether. We have evaluated an alternative sectioning protocol for stabilizing the tissue microarray surface by placing an adhesive tape “window” over the face of the paraffin block prior to sectioning. Once sectioned, the tape/sections are transferred directly onto coated microscope slides, thereby avoiding routine floating of sections on a water bath. After sectioning with either the tape transfer or standard protocols, slides were stained either using hematoxylin and eosin or immunohistochemistry using antibodies to S-100 protein and the tissue specific antigens, keratin (AE1/3) and the leukocyte common antigen CD45. We found that the tape method produced thicker sections that were darker and more densely packed with loss of tissue definition compared to sections prepared using water bath flotation. Quantitative image analysis of immunohistochemical staining demonstrated that the tape method produced a higher incidence of nonspecific staining, which raised the potential for false positive staining.     

Identification of leucocyte surface antigens in paraffin- embedded bovine tissues using a modified formalin dichromate fixation

A modified fixative of formalin dichromate was combined with a cold embedding procedure for the preservation of bovine leucocyte surface antigens. Fourteen monoclonal antibodies recognizing seven bovine leucocyte surface antigens (BoCD1w2, BoCD4, BoCD8, BoWC1, BoWC3, BoWC4 and BoIgM) were applied as primary antisera in a sensitive avidin--biotin--peroxidase complex detection method. The staining results were compared with those obtained in cryostat and routinely formalin-fixed sections of corresponding tissue samples. Using the modified formalin dichromate fixative and the cold embedding procedure, all the leucocyte surface antigens tested were detectable immunohistologically in paraffin sections with a generally more distinct staining than in traditionally processed tissues. Morphological structures were better preserved than in cryostat sections but, to some extent, were poorer when compared with routinely formalin-fixed tissues. However, this method suggests that there are only mild masking effects and provides an alternative to the use of unfixed material, particularly for morphological--immunohistochemical investigations     

Effects of fixation and processing on the immunohistochemical visualization of type-I, -III and -IV collagen in paraffin-embedded liver tissue

We compared the effects of different fixatives and enzymatic-digestion procedures on the immunohistochemical visualization of type-I, -III and -IV collagen in paraffin-embedded normal human liver sections. None of the fixatives tested allowed the staining of these antigens without prior enzymatic digestion. The best results i.e. strong staining intensity and well-defined localization, were obtained when liver tissue was fixed in Bouin's fluid or in other solutions containing picric acid. Several other fixatives, including Carnoy's fluid, Lillie's AAF, 10% neutral formalin and 96% ethanol, gave unsatisfactory results. Pepsin was ineffective for unmasking type-I and -III collagen antigens, and was only partially effective for visualizing the type-IV collagen antigen. The best results were obtained when material fixed in Bouin's fluid was embedded in paraffin and digested with trypsin. Using this procedure, the results were comparable to those obtained in unfixed frozen sections with respect to the staining intensity, specificity and non-specific staining.     

Variability of laminin immunoreactivity in human autopsy brain

Summary Laminin immunoreactivity is thought to be masked in formalin-fixed sections since proteolytic treatment is required to unmask it. We analyzed this masking with frozen and formalin-fixed human autopsy brains obtained at various postmortem periods. In unfixed, frozen sections, intense immunoreactivity was invariably detected in vascular walls of entire sections. When such sections were postfixed in formalin, immunoreactivity was not diminished even after prolonged fixation. In vibratome sections of brain fixed in formalin in situ, immunoreactivity varied with postmortem delay: in most cases, immunoreactivity was weak and restricted to superficial cortical layers. However, the extent of immunoreactivity increased with postmortem delay. Two cases fixed after prolonged postmortem periods revealed moderate immunoreactivity throughout the sections. We also investigated rat brains processed without postmortem delay. In unfixed frozen sections, immunoreactivity again was observed throughout the sections, independent of the length of any postfixation. In vibratome sections of fixed rat brain, immunoreactivity was restricted to the cutting margins of the brain blocks and around a trauma-induced cortical lesion, regardless of how long the blocks had been kept in fixative. Our data suggest that postmortem proteolysis accomplishes similar unmasking of laminin antigen as digestion on paraffin sections and that such unmasking can also be effected by proteolysis induced by damaging tissue during cryostat sectioning of fresh tissue.     

Histopathologic Technique for the Morphological Examination of Fixed or Frozen Solid Tumor Cells in Soft Agar

Two simple techniques are described for preparing sections from soft agar colony cultures of tumor cells. Tumor cells grown in soft agar can be frozen, sectioned, and stained and/or fixed in formalin, embedded in paraffin, sectioned, mounted on glass slides, and stained. The methods are simple and reproducible. These cells can be stained with various stains and the staining quality is excellent. The paraffin blocks and microscope slides can be stored for permanent record. The use of these techniques should provide better understanding of the histomorphologic characteristics of neoplastic cells which grow in soft agar and should expand and refine prognosis and diagnosis of malignant tumors.     

Enzyme histochemical discrimination between tryptase and chymase in mast cells of human gut

We tested four synthetic substances for their histochemical value to demonstrate the catalytic activities of chymase or tryptase in mast cells in sections of human gut. Both Suc-Ala-Ala-Phe-4 methoxy-2-naphthylamide (MNA) and N-acetyl-L-methionine-alpha-naphthyl ester (alpha-N-O-Met) reacted with chymase but not tryptase in mast cells. Conversely, D-Val-Leu-Arg-MNA and Z-Ala-Ala-Lys-MNA were hydrolyzed by mast cell tryptase but not chymase. These results were confirmed by use of two inhibitors of chymotrypsin-like activity, chymostatin and Z-Gly-Leu-Phe-chloromethyl ketone (CK) and two inhibitors of trypsin-like activity, Tos-Lys-CK and D-Val-Leu-Arg-CK. Excellent staining reactions were obtained on cryostat sections of unfixed or aldehyde-fixed tissues and on paraffin sections of Carnoy-fixed tissues. For chymase, however, Suc-Ala-Ala-Phe-MNA is preferred on cryostat sections because it is more specific. On paraffin sections alpha-N-O-Met is preferred because other cells are not then stained. For tryptase, Z-Ala-Ala-Lys-MNA was more selective and more specific and is the preferred general purpose substrate on cryostat sections of aldehyde-fixed tissues and for paraffin sections. D-Val-Leu-Arg-MNA is the preferred substrate for cryostat sections of unfixed tissue. Only a limited number of mast cells showed a reaction for chymase, and these occurred mainly in the submucosa. All mast cells, however, gave a reaction for tryptase, and we recommend the use of either substrate for this enzyme for routine detection of mast cells in human tissues. Double staining for the two main mast cell proteases is most conveniently undertaken on paraffin sections of Carnoy-fixed tissues using MNA substrates for tryptase and alpha-N-O-Met for chymase.     

Comparative study of procedures for histological detection of lectin binding by use of Griffonia simplicifolia agglutinin I and gastrointestinal mucosa of the rat

The histological localisation of alpha-D-galactopyranosyl residues in glycoconjugates of rat stomach and duodenal mucosae was studied by use of Griffonia simplicifolia agglutinin I, i.e. the isolectin mixture (A + B) and the isolectin B4 (B4). Cryostat sections which were either unfixed or acetone fixed and paraffin sections from both ethanol-acetic acid and formaldehyde fixed tissue blocks were compared. Cellular details were better preserved in paraffin than in cryostat sections. Reactivity of cells binding GS I was less sensitive after formaldehyde than after ethanol-acetic acid fixation inasmuch as higher concentrations of lectins were needed. This drawback could be overcome by trypsinisation of the sections. The binding pattern of GS I (A + B) corresponded with that of GS I (B4) in either cryostat or paraffin sections. GS I was detected in the cytoplasm of parietal cells and in Brunner's gland cells. In duodenal crypts and villi, lectin was bound to supranuclear regions in the cytoplasm of columnar and goblet cells. The staining efficiency of fluorescein (FITC), horseradish peroxidase (HRP) and colloidal gold particle (CGP) labels in both direct and indirect lectin stainings was compared. Under all experimental conditions, indirect methods required lower concentrations of lectins than direct ones; indirect procedures increased sensitivity about 5-10 fold. CGP labels were always of highest sensitivity when gold particles were further developed by a silver precipitation method. HRP was not as efficient in lectin localisation as CGP, but cytochemical staining was more convenient in routine work. Direct FITC labellings proved to be of lowest sensitivity.     

Immunohistology on formol-sublimate fixed and paraplast embedded kidney tissue with comparison to formalin fixation and to pre-embedding immunofluorescent staining

Many alternative methods for immunopathological evaluation of kidney tissue are now available. Immunofluorescent or immunoperoxidase staining of kidney can be performed after formalin fixation and paraffin embedding. This is also possible after fixation with formol-sublimate (Stieve's fluid) using the immunoperoxidase technique or by immunofluorescence after removal of mercury. Reduction of strong nonspecific fluorescence caused by the mercury fixative parallels the elimination of mercury as verified by X-ray microanalysis of the sections. Using a mouse model with injection of graded dilutions of antiglomerular basement membrane antibodies, immunofluorescent staining after Stieve fixation and embedding in Paraplast was about 60% of that in cryostat sections. Immunofluorescent staining after mercury removal can be followed by silver staining for detailed morphologic study of the same 1 micron Paraplast sections. A case of antiglomerular basement membrane glomerulonephritis is illustrated in more detail to show the necessity of alternative methods, including the technique presented, pre-embedding immunofluorescent staining of Epon sections, and electron microscopy, to make a reliable diagnosis of this disease.     

ULTRASTRUCTURAL LOCALIZATION OF ACID MUCOSUBSTANCES IN THE MOUSE COLON WITH IRON-CONTAINING STAINS

Selective ultrastructural staining of acid mucosubstances in sites containing histochemically identifiable sulfo- and sialomucins has been obtained in fixed cryostat sections with both ferric chloride and colloidal iron solutions. The rectosigmoid region of mouse colon was fixed in glutaraldehyde, formalin, or phosphate-buffered osmium tetroxide, and 40 µ cryostat sections of this material were treated with 0.1 to 0.4% ferric chloride or with a solution of dialyzed ferric chloride, ammonia, and glycerin. Specific staining depended upon the pH of the iron-containing solutions, and the optimal value was found to be approximately 2.0. Specific localization of acid mucosubstances has been noted in intracellular sites, including globules within colonic goblet cells and "deep crypt" mucous cells, small vesicles of the superficial nongoblet epithelial cells, and Golgi lamellae within each of these cell types. Extracellular material, presumed to be acid mucosubstance, was found on the surface of the epithelial microvilli and on the lumenal surface of capillary endothelium. Similar material formed a reticular network surrounding stromal cells, collagen bundles, and various colonic connective tissue elements.     

Variability of laminin immunoreactivity in human autopsy brain.

Laminin immunoreactivity is thought to be masked in formalin-fixed sections since proteolytic treatment is required to unmask it. We analyzed this masking with frozen and formalin-fixed human autopsy brains obtained at various postmortem periods. In unfixed, frozen sections, intense immunoreactivity was invariably detected in vascular walls of entire sections. When such sections were postfixed in formalin, immunoreactivity was not diminished even after prolonged fixation. In vibratome sections of brain fixed in formalin in situ, immunoreactivity varied with postmortem delay: in most cases, immunoreactivity was weak and restricted to superficial cortical layers. However, the extent of immunoreactivity increased with postmortem delay. Two cases fixed after prolonged postmortem periods revealed moderate immunoreactivity throughout the sections. We also investigated rat brains processed without postmortem delay. In unfixed frozen sections, immunoreactivity again was observed throughout the sections, independent of the length of any postfixation. In vibratome sections of fixed rat brain, immunoreactivity was restricted to the cutting margins of the brain blocks and around a trauma-induced cortical lesion, regardless of how long the blocks had been kept in fixative. Our data suggest that postmortem proteolysis accomplishes similar unmasking of laminin antigen as digestion on paraffin sections and that such unmasking can also be effected by proteolysis induced by damaging tissue during cryostat sectioning of fresh tissue.     

Concentrated Formalin Versus A 10% Solution as a Fixative Preceding Silver Staining

The effect of concentrated and 10% formalin as fixatives for the silver staining of axons in the central nervous system in a series of mammals has been compared. It is felt that concentrated formalin constitutes a better fixative for this purpose in several respects: it has a greater speed of penetration without causing additional distortion or artifacts, the staining time can be reduced somewhat and the normal configuration of the axons is better preserved particularly in the largest ones. Satisfactory results were obtained following 51/2hours fixation in concentrated formalin. Further, concentrated formalin is at least equal to a 10% solution in the ease in which paraffin and frozen blocks can be sectioned, in the clearness of the background of stained sections and in the consistency of results.     

Immunohistochemical Double Staining of Microwave Enhanced and Nonenhanced Nuclear and Cytoplasmic Antigens

Many of the antigens commonly investigated in histopathology can be enhanced by microwave pretreatment (MWPT) of formalin fixed, paraffin embedded tissue sections. We developed a double labeling method using microwave heating to detect otherwise undetectable nuclear antigens combined.with immunohisto-chemistry (IHC) of cytoplasmic or membranous antigens that do not benefit from MWPT. We used the same primary antibody solutions used in single antibody IHC. The staining technique is based on the alkaline phosphatase anti-alkaline phosphatase (APAAP) and the labeled avidin-biotin (LSAB) methods. Four different protocols were tested, each modifying the sequence of MWPT, APAAP and LSAB staining. In this study Ki67, estrogen receptor, progesterone receptor, c-neu, CD68 and desmin primary antibodies were used in routinely formalin fixed, paraffin embedded tissues of 50 tumor specimens. MWPT followed by LSAB for microwave enhanced antigens and APAAP for antigens that cannot be enhanced by MWPT gave the best double staining results. This method improves characterization of tumor cell features from paraffin embedded tissue and should aid analysis of tumor differentiation, receptor status and nuclear proteins in the single cells in archival tissues.     

Acceleration of the Movat Pentachrome One Stain by Heat

The conventional staining time for Movat's pentachrome I stain (Arch. Path., 60: 289-295, 1955) was shortened from about 18-19 hr to about 2.5 hr. The ammoniated alcohol and the resorcin-fuchsin staining baths were heated to 56 C. All other steps in the technic were performed at 25-27 C. Staining properties of paraffin sections of many types of tissue fixed in formalin, formol-sublimate-acetic, or in Bouin's fluid, showed that staining with resorcin-fuchsin at the elevated temperature gave the same results as staining at room temperature.