Controlled Differentiation of Cells and Connective Tissue Fibers in Silver Staining

Controlled silver staining of connective tissue fibers and sometimes of these fibers and cells simultaneously can be obtained. 1. Fix in 10% formalin. Embed in paraffin and cut sections as usual, but do not mount them on slides. Deparaffinize and hydrate through xylene, alcohols and distilled water and henceforth treat them the same as frozen sections. Real frozen sections can also be used. 2. Treat with a freshly prepared 1% solution of KMnO₄, usually 15-60 sec, sometimes up to 10 min. 3. Wash in distilled water, 5-10 sec. 4. Decolorize in 2% potassium metabisulfite, 10-20 sec. 5. Place in distilled water, 1 min. 6. Sensitize with 2% iron alum, 1 min. 7. Place in distilled water, 1 min. 8. Impregnate in Gomori's silver oxide solution, 2 min. 9. Wash in a 1.5% aqueous solution of pyridine, about 15 sec. 10. Reduce in a mixture containing 0.25% gelatin and 2% formalin 1 min. 11. Repeat steps 7 to 10 once or several times until the connective tissue fibers are completely stained. For cell staining (which may fail) proceed as follows: After the first insufficient staining of the connective tissue fibers, rinse in distilled water, dip for 1 sec in Gomori's solution and reduce immediately in gelatin-formalin without previous washing in pyridined water. This step can be repeated. 12. If the staining is too strong, decolorize as needed in 2% iron alum. 13. Toning in 0.2% gold chloride, 5 min or more, followed by fixation in 5% sodium thiosulfate, 1 min, is optional. Counterstain as desired. 14. Wash in tap water, dehydrate, clear in xylene and mount in balsam. The same technique applied to sections attached to slides gives good results but inferior to that obtained in paraffin sections processed in the loose, unmounted condition.     

Effects of Ph on Post-Mortem Retention of Trypan Blue Vital Staining in Tissues of Mice

Vital staining of aortas from mice injected subcutaneously (daily for 5 days) with trypan blue was studied. In routine paraffin sections elastic membranes were observed to be well stained and other medial elements unstained following fixation in 10% formaldehyde (25% formalin) at pH 7-9. An identical pattern of vital staining was observed in specimens that had been immersed for 48 hr in saline solutions at pH 7-11. Elastic membranes were not stained, but intermembranous connective tissue was stained after the following: (1) fixation in 10% formaldehyde at pH 1-4 and in Lavdowsky's solution (ethanol, formaldehyde, water and glacial acetic acid), pH 2.3-2.8; and (2) immersion in saline for 48 hr at pH 14. Aortic elastic membranes were vitally stained after fixation by intracardiac perfusion with 10% formaldehyde (pH 7-8) but not after perhion with Lavdowsky's fixative (pH 2.3-2.8). Vital staining was limited to medial elastic membranes in sections of fresh aorta made in a cryostat or by a regular freezing microtome. The vital staining (coarse cytoplasmic granules of dye) within macrophages (Kupffer cells and others) and in cytoplasm of renal tubular epithelium was well demonstrated following use of all methods discussed above     

Histological assessment of PAXgene tissue fixation and stabilization reagents

Within SPIDIA, an EC FP7 project aimed to improve pre analytic procedures, the PAXgene Tissue System (PAXgene), was designed to improve tissue quality for parallel molecular and morphological analysis. Within the SPIDIA project promising results were found in both genomic and proteomic experiments with PAXgene-fixed and paraffin embedded tissue derived biomolecules. But, for this technology to be accepted for use in both clinical and basic research, it is essential that its adequacy for preserving morphology and antigenicity is validated relative to formalin fixation. It is our aim to assess the suitability of PAXgene tissue fixation for (immuno)histological methods. Normal human tissue specimens (n = 70) were collected and divided into equal parts for fixation either with formalin or PAXgene. Sections of the obtained paraffin-embedded tissue were cut and stained. Morphological aspects of PAXgene-fixed tissue were described and also scored relative to formalin-fixed tissue. Performance of PAXgene-fixed tissue in immunohistochemical and in situ hybridization assays was also assessed relative to the corresponding formalin-fixed tissues. Morphology of PAXgene-fixed paraffin embedded tissue was well preserved and deemed adequate for diagnostics in most cases. Some antigens in PAXgene-fixed and paraffin embedded sections were detectable without the need for antigen retrieval, while others were detected using standard, formalin fixation based, immunohistochemistry protocols. Comparable results were obtained with in situ hybridization and histochemical stains. Basically all assessed histological techniques were found to be applicable to PAXgene-fixed and paraffin embedded tissue. In general results obtained with PAXgene-fixed tissue are comparable to those of formalin-fixed tissue. Compromises made in morphology can be called minor compared to the advantages in the molecular pathology possibilities.     

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

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 microns in thickness) which were prepared in ten different ways: fixed by alcohol or formalin; stained by hematoxylin-eosin (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.     

Iron Gallein Elastic Method—A Substitute for Verhoeff'S Elastic Tissue Stain

A method for staining elastic fibers in formalin fixed, paraffin embedded sections is described. After deparaffinizing and dehydration. sections are stained for 30 minutes in a solution prepared by mixing equal parts of 1% gallein dissolved in ethylene glycol and absolute alcohol (1:4), and 1.16% aqueous ferric chloride in 1% hydrochloric acid. The sections are washed in water and then differentiated in 2% ferric chloride for 2 minutes. After washing in water, the sections am counterstained with a variant of Van Girson's picric acid-acid fuchsin for 1 minute. The results are similar to Verhoeff s elastic stain with elastic fibers staining black. An advantage to this staining procedure is that visually controlled differentiation is not necessary.     

Iron gallein elastic method---a substitute for Verhoeff's elastic tissue stain.

A method for staining elastic fibers in formalin fixed, paraffin embedded sections is described. After deparaffinizing and dehydration, sections are stained for 30 minutes in a solution prepared by mixing equal parts of 1% gallein dissolved in ethylene glycol and absolute alcohol (1:4), and 1.16% aqueous ferric chloride in 1% hydrochloric acid. The sections are washed in water and then differentiated in 2% ferric chloride for 2 minutes. After washing in water, the sections are counterstained with a variant of Van Gieson's picric acid-acid fuchsin for 1 minute. The results are similar to Verhoeff's elastic stain with elastic fibers staining black. An advantage to this staining procedure is that visually controlled differentiation is not necessary.     

Studies on Bovine Mast Cells: I. The Effect of Formalin Fixation

Studies are reported on the effect of aqueous formalin fixation on the bovine mast cell. Paraffin sections of bovine skin were prepared from tissues fixed for 7 to 72 hr in 10% neutral formalin in saline, and from tissues which were treated with water for various times both before and after formalin fixation. Metachromatic halos, scattering of the granules, and fraying of the cell outline, were seen in the mast cells of tissues washed in water before fixation. Exposure to water after fixation did not produce these artifacts. The tendency towards orthochromatic staining, and the occurrence of perinuclear clear zones are probably effects of the formalin and not of overstaining or of exposure of the tissue to water. The majority of the metachromatic material in the bovine mast cell is water soluble, and may be removed by washing in water before fixation, whereas the granules are relatively resistant to water. The optimum time of formalin fixation of bovine skin to permit studies on the metachromatic material of the mast cells was 24 to 36 hr.     

Demonstration of laminin, a basement membrane glycoprotein, in routinely processed formalin-fixed human tissues

Laminin was demonstrated by immunoperoxidase and immunofluorescence staining in sections of normal human tissues fixed in formalin and routinely processed in paraffin. Exposure of the sections to a solution of pepsin (Burns et al. (1980) Histochemistry 67:73-78) revealed the antigenicity of this basement membrane glycoprotein. Sections from paraffin blocks stored for years at room temperature could be stained with this procedure. Normal human tissues, developing fetal tissues and tumors could be stained with this method. The staining patterns were similar to those seen in unfixed frozen sections. It thus appears that basement membrane components can be detected by immunohistological means from routinely processed histological samples, once the sections are pretreated with proteases. Staining for laminin could be used in embryonic studies and in histopathology to study the relation of cells to basement membranes and for the visualization of normal and abnormal vascularization.     

Block Staining of Mammalian Tissues with Hematoxylin and Eosin

Various mammalian tissues were stained en bloc with hematoxylin and eosin after fixation and prior to embedding in paraffin wax and sectioning. The choice of fixative is important and best results are obtained using Worcester's Fluid, a combination of saturated aqueous mercuric chloride, formaldehyde, and glacial acetic acid. After fixation, blocks of tissue up to 1.5 cm thick are stained for seven days in hematoxylin. Excess stain is removed by washing tissues in running water overnight. Tissue blocks then are dehydrated with graded concentrations of ethyl alcohols to 80% and counterstained, with further dehydration, in 0.5% spirit soluble eosin in 90% ethyl alcohol for five days. The tissue is subsequently transferred to 90% ethyl alcohol overnight to differentiate eosin staining; dehydration is completed in absolute ethyl alcohol. The blocks are cleared in cedarwood oil and briefly in xylene prior to embedding, sectioning, and mounting. Following removal of wax by xylene, coverslips are applied.

General morphological and histological features were particularly well differentiated and very selectively and reliably stained by this method.     

Block staining of mammalian tissues with hematoxylin and eosin

Various mammalian tissues were stained en bloc with hematoxylin and eosin after fixation and prior to embedding in paraffin wax and sectioning. The choice of fixative is important and best results are obtained using Worcester's Fluid, a combination of saturated aqueous mercuric chloride, formaldehyde, and glacial acetic acid. After fixation, blocks of tissue up to 1.5 cm thick are stained for seven days in hematoxylin. Excess stain is removed by washing tissues in running water overnight. Tissue blocks then are dehydrated with graded concentrations of ethyl alcohols to 80% and counterstained, with further dehydration, in 0.5% spirit soluble eosin in 90% ethyl alcohol for five days. The tissue is subsequently transferred to 90% ethyl alcohol overnight to differentiate eosin staining; dehydration is completed in absolute ethyl alcohol. The blocks are cleared in in cedarwood oil and briefly in xylene prior to embedding, sectioning, and mounting. Following removal of wax by xylene, coverslips are applied. General morphological and histological features were particularly well differentiated and very selectively and reliably stained by this method.     

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.     

Identification in Histological Sections of Species Origin of Cells from Mouse, Rat and Human

A simple method for identifying the species of origin of mammalian cells in tissue sections using Hoechst dye #33258 is described. This rapid procedure involves staining fresh frozen or formalin fixed paraffin sections with 4 µg/ml of Hoechst #33258 for one minute at room temperature; following one to five minutes of washing in running tap water, the slides are coverslipped using McIlvaine's buffer (pH 5.5) and sealed with clear lacquer. Sections examined by fluorescence microscopy indicate that mouse cells contain several small discrete intranuclear fluorescent bodies, which are absent in cells from either rat or human. This simple technique should be useful in studying developmental phenomena in chimeric organs.     

Differentiation of Myofibrillae, Reticular and Collagenous Fibrils in Vertebrates

A procedure for the differentiation of the mesenchymal derivatives, myofibrillae, reticular and collagenous fibers is presented. Formol-Zenker fixation (5-12 hours) is followed by the washing, iodinization, dehydration and paraffin embedding steps routine for that fixative with the following modifications. Zirkle's butyl alcohol series is used for dehydration and infiltration with paraffin as well as in the alcohol slide series. Embedding paraffin used is Parawax plus 8-10% bayberry wax. Tissue-exposed surface of paraffin block is soaked in water overnight before cutting serial sections at 3-5μ. Sections are mounted using the dilute albumen method, and the slides, thoroughly dried at 37^oC. overnight, are left at 60^o for 10 minutes to melt the paraffin of the sections. Before staining, the sections are given a preliminary treatment with potassium permanganate and oxalic acid. For reticular staining a 10% silver nitrate bath is succeeded by an ammoniacal silver carbonate solution followed by reduction in 1% neutral formalin, toning in gold chloride and fixing in sodium thiosulphate. Myofibrillae, the sacroplasmic limiting membrane and other sarcous elements are stained by Heidenhain's azocarmine solution, adult tissues at room temperature and fetal tissues at 50 ^oC. Differentiation in phosphotungstic acid is followed by the staining of collagenous fibers. For adult tissue, light green SF (C.C.) is used and for fetal tissue, fast green FCF (C.C). A discussion of the preparation of ammoniacal silver solutions is included. Both stock and used solutions of ammoniacal silver have been in use by the author for over a period of two years.     

A comparison of fixation methods on lung morphology in a murine model of emphysema

Emphysema is characterized by enlargement of the alveoli, which is the most important parameter to assess the presence and severity of this disease. Alveolar enlargement is primarily defined on morphological criteria; therefore, characterization of this disease with morphological parameters is a prerequisite to study the pathogenesis. For this purpose, different methods of lung fixation were evaluated in a murine model of LPS-induced lung emphysema. Five different methods of lung fixation were evaluated: intratracheal instillation of fixatives, in situ fixation, fixed-volume fixation, vascular whole body perfusion, and vacuum inflation. In addition, the effects of three different fixatives (10% formalin, Carnoy's, and agarose/10% formalin solution) and two embedding methods (paraffin and plastic) were investigated on the murine lung morphology. Mice received intranasal administration of LPS to induce alveolar wall destruction. Quantification of air space enlargement was determined by mean linear intercept analysis, and the histological sections were analyzed for the most optimal fixation method. Additionally, routine immunohistological staining was performed on lung tissue of PBS-treated mice. Intratracheal instillation of formalin or agarose/formalin solution, in situ fixation, and fixed-volume fixation provided a normal lung architecture, in contrast to the lungs fixed via whole body perfusion and vacuum inflation. Formalin-fixed lungs resulted in the most optimal lung morphology for lung emphysema analysis when embedded in paraffin, while for Carnoy's fixed lungs, plastic embedding was preferred. The histological findings, the mean linear intercept measurement, and the immunohistochemistry data demonstrated that fixation by intratracheal instillation of 10% formalin or in situ fixation with 10% formalin are the two most optimal methods to fix lungs for alveolar enlargement analysis to study lung emphysema.     

Staining Nerve Fibers After Sublimate-Acetic and After Bouin'S Fluid

A silver staining method for paraffin sections of material fixed in HgCl₂, sat. aq., with 5% acetic acid is as follows. Process the sections through the usual sequence of reagents, and including I-KI in 70% alcohol, thiosulfate (5% aq.), washing and back to 70% alcohol containing 5% of NH₄OH (conc. aq.). After 3 minutes in the ammoniated alcohol, wash through tap water and 2 changes of distilled water and silver 5-10 minutes at 25°C. in 15% AgNO₃ aq. to which 0.02 ml. of pyridine per 100 ml. has been added. Blot the slide, but not the section and do not rinse. Reduce at 45°C. in 0.1% pyrogallol in 55% alcohol, then rinse in 55% alcohol and wash in water. The remainder of the process consists of gold toning, intensifying in oxalic acid, fixing in 5% Na₂S₂O₃, washing, dehydrating, clearing and covering. When the specimen contains much smooth muscle, the I-KI solution is acidified before use by adding 2 ml. of 1N nitric acid per 100 ml., and the sections treated for 3 minutes instead of the usual 2 minutes. Formalin should not be added to sublimate-acetic, but specimens that do not contain strongly argyrophilic nonneural tissue may be fixed in formalin or, preferably, Bouin's fluid. Sections of tissue after the latter type of fixation will not require the I-KI and thiosulfate but can go from 95% alcohol to the ammoniated alcohol. The advantages of fixing in HgCl₂-acetic acid are suppression of the staining of connective tissue and intensifying the staining of nerve fibers.     

Demonstration of laminin,a basement membrane glycoprotein,in routinely processed formalin-fixed human tissues

Summary Laminin was demonstrated by immunoperoxidase and immunofluorescence staining in sections of normal human tissues fixed in formalin and routinely processed in paraffin. Exposure of the sections to a solution of pepsin (Burns et al. (1980) Histochemistry 6773–78) revealed the antigenicity of this basement membrane glycoprotein. Sections from paraffin blocks stored for years at room temperature could be stained with this procedure. Normal human tissues, developing fetal tissues and tumors could be stained with this method. The staining patterns were similar to those seen in unfixed frozen sections. It thus appears that basement membrane components can be detected by immunohistological means from routinely processed histological samples, once the sections are pretreated with proteases. Staining for laminin could be used in embryonic studies and in histopathology to study the relation of cells to basement membranes and for the visualization of normal and abnormal vascularization.To whom offprint requests should be sent     

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.     

Immunofluorescent Staining of Leptospires in Pepsin Treated Histologic Sections

A standard immunofluorescent method was modified for the staining of leptospires in formalin fixed, paraffin embedded tissues. Routine histologic sections were deparaffinized and treated with pepsin prior to staining. Pepsin treatment greatly enhanced subsequent staining of leptospires in naturally infected bovine and porcine tissues as well as in artificially infected tissues. Leptospires in naturally infected bovine tissues were usually undetectable in untreated sections but clearly visible in stained pepsin-treated sections. Naturally infected porcine kidney usually contained high levels of leptospiral antigen which could be stained without prior pepsin treatment. However, pepsin treatment of porcine tissues greatly increased the amount of leptospiral antigen detectable and made individual leptospires more conspicuous. The staining method could employ a single antiserum for the staining of leptospires from 13 serogroups. Also, leptospires could be stained in tissues stored in formalin for more than 14 months and in 26-year-old paraffin embedded tissues.     

The Demonstration of Beta Cells in Pancreatic Islets and Their Tumors

The stain is applied routinely to tissues fixed in 10% buffered formalin (pH near 7.0) or in Bouin's fluid. Bring paraffin section to water as usual and mordant 72 hr in 5% CrCl₃ dissolved in 5% acetic acid. Wash in water and in 70% alcohol and stain 6 hr. Formula of staining solution: new fuchsin, 1% in 70% alcohol, 100 ml; HCl, conc., 2 ml and paraldehyde, 2 ml, mixed together and added to the dye solution; let stand 24 hr before use. After staining, wash in running tap water 5-10 min, rinse in distilled water and counterstain if desired. Dehydration in alcohol, clearing and covering completes the process. When the paraldehyde is obtained from a freshly opened bottle, standardized staining times can be used and thus eliminate the necessity of differentiating individual slides. The granules of beta cells stained deep blue to purple and were demonstrated in the pancreatic islet of man, dog, mouse, frog, guinea pig and rabbit.     

Demonstration of Degenerating Nerve Fibers by a Modified Cajal Technic

Whole brains of cat were fixed in two changes of cold acetone (24 hours each) and embedded directly in paraffin. The degeneration time recommended is 5 days. Mounted sections 15-20 μ thick were deparaffined, washed in absolute alcohol and given successive treatments of 6 hours each with 1% ammoniated absolute alcohol and pure pyridine, washing well with distilled water between them and after the pyridine. Impregnation in 2% silver nitrate 12 hours at 30°C., rinsing in absolute alcohol and reducing in a 95% alcoholic solution of pyrogallol and formalin (3% and 5%) was followed by 50% alcohol, thorough washing in distilled water, toning in 1% gold chloride and intensification in 1% oxalic acid. Treatment in 10% sodium thiosulfate solution, washing, dehydrating and covering completed the procedure. Normal fibers, degenerating fibers and terminals were stained specifically.