The importance of fixation procedures on DNA template and its suitability for solution-phase polymerase chain reaction and PCR in situ hybridization

Summary Conventional solution-phase polymerase chain reaction (PCR) and in situ PCR/PCR in situ hybridization are powerful tools for retrospective analysis of fixed paraffin wax-embedded material. Amplification failure using these techniques is now encountered in some centres using archival fixed tissues. Such ailures may not only be due to absent target DNA sequences in the tissues, but may be a direct effect of the type of fixative, fixation time and/or fixation temperature used. The type of nucleic acid extraction procedure applied will also influence amplification results. This is particularly true with in situ PCR/PCR in situ hybridization.To examine these effects in solution-phase PCR, -globin gene was amplified in 100 mg pieces of tonsillar tissue fixed in Formal saline, 10% formalin, neutral buffered formaldehyde, Carnoy's, Bouin's, buffered formaldehyde sublimate, Zenker's, Helly's and glutaraldehyde at 0 to 4°C, room temperature and 37°C fixation temperatures and for fixation periods of 6, 24, 48 and 72 hours and 1 week. DNA extraction procedures used were simple boiling and 5 days' proteinase K digestion at 37°C. Amplified product was visible primarily yet variably from tissue fixed in neutral buffered formaldehyde and Carnoy's, whereas fixation in mercuric chloride-based fixatives produced consistently negative results. Room temperature and 37°C fixation temperature appeared most conducive to yielding amplifiable DNA template. Fixation times of 24 and 48 hours in neutral buffered formaldehyde and Carnoy's again favoured amplification.Fixed SiHa cells (containing 1–2 copies of HPV 16) were examined using PCR in situ hybridization for the amplification of HPV 16. Discrete and diffuse amplification signals were obtained. Neutral buffered formaldehyde fixation for 12–24 hours yielded amplifiable material suitable for use with PCR in situ hybridization. Overall amplification success within cellular preparations was 40%, with non-specific background staining also seen. Possible technical problems encountered with PCR in situ hybridization are discussed.     

Staining of interphase nuclei and mitotic figures in cultured cells with alcian blue 8GX

Cultured endothelial cells derived from bovine calf pulmonary artery were subjected to a variety of fixatives and stained with 1% Alcian blue 8GX at pH 2.59 to 3.26. Within this range of pH, interphase nuclei and especially mitotic figures were (a) strongly stained in cells fixed with 10% formalin (phosphate buffered or unbuffered) or 2.5% buffered glutaraldehyde, (b) weakly stained or unstained in cells fixed in formaldehyde containing divalent cations, and (c) unstained in cells fixed in acetic acid-containing fluids. However, optimal nuclear staining with Alcian blue under the conditions of this study was judged to be achieved after fixation with neutral phosphate buffered 10% formalin. Endothelial cell cytoplasm exhibited a similar fixative-dependent staining. At pH 2.59 the cytoplasm of interphase cells fixed in formaldehyde (containing no divalent cations) or glutaraldehyde remained unstained; however, at higher pH cytoplasmic staining did occur and it increased as pH increased. In contrast, when these latter fixatives were employed the cytoplasm of mitotic cells stained at all pH levels tested. In cultured endothelial cells after appropriate fixation, 1% Alcian blue 8GX (pH 2.59) was found to possess the ability to stain nuclei with a selectivity and intensity that compared favorably to those of the Feulgen reaction of Heidenhain iron hematoxylin but without the latters' length and complexity. Therefore, this procedure may provide a rapid, simple, and selective method for visualizing interphase nuclei or mitotic figures, or both in the majority of cultured cells.     

Staining of interphase nuclei and mitotic figures in cultured cells with Alcian blue 8GX

Summary Cultured endothelial cells derived from bovine calf pulmonary artery were subjected to a variety of fixatives and stained with 1% Alcian blue 8GX at pH 2.59 to 3.26. Within this range of pH, interphase nuclei and especially mitotic figures were (a) strongly stained in cells fixed with 10% formalin (phosphate buffered or unbuffered) or 2.5% buffered glutaraldehyde, (b) weakly stained or unstained in cells fixed in formaldehyde containing divalent cations, and (c) unstained in cells fixed in acetic acid-containing fluids. However, optimal nuclear staining with Alcian blue under the conditions of this study was judged to be achieved after fixation with neutral phosphate buffered 10% formalin. Endothelial cell cytoplasm exhibited a similar fixative-dependent staining. At pH 2.59 the cytoplasm of interphase cells fixed in formaldehyde (containing no divalent cations) or glutaraldehyde remained unstained; however, at higher pH cytoplasmic staining did occur and it increased as pH increased. In contrast, when these latter fixatives were employed the cytoplasm of mitotic cells stained at all pH levels tested. In cultured endothelial cells after appropriate fixation, 1% Alcian blue 8GX (pH 2.59) was found to possess the ability to stain nuclei with a selectivity and intensity that compared favorably to those of the Feulgen reaction or Heidenhain iron hematoxylin but without the latters’ length and complexity. Therefore, this procedure may provide a rapid, simple, and selective method for visualizing interphase nuclei or mitotic figures, or both in the majority of cultured cells.     

The effect of formaldehyde fixation and dehydration on ox adrenal medulla with respect to the chromaffin reaction and post-chroming

Summary Chromaffin tissue was fixed in 4% formaldehyde buffered to pH 5.8 and subsequently dehydrated. The fixing fluid and alcohols were examined for catechol amine and adenosine nucleotide loss by paper and columnar chromatography. Much of these substances was found to escape during fixation and dehydration. The quantitative loss of ATP and catechol amines during formaldehyde fixation was shown to be 30–40%. Dehydration increased the loss to approximately 90%. The relevance of these results to the chromaffin reaction and post-chroming were discussed.     

The effects of mercaptoethanol-formaldehyde on tissue fixation and protein retention

Summary The study compared the effects of mercaptoethanol-formaldehyde and formaldehyde alone, on tissue fixation and protein retention in human and mouse tissues. Shrinkage of tissues and the penetration rate of the fixatives were assessed. The cross-linking ability of the fixatives was determined by viscometry, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and spectrophotometry, using bovine serum albumin and human haemoglobin. Tissues fixed in buffered 0.0025% mercaptoethanol-4% formaldehyde showed good nuclear and cytoplasmic detail, better than those fixed in buffered 4% formaldehyde. There was no significant difference in shrinkage. A mixture of 0.0025% mercaptoethanol-4% formaldehyde penetrated faster into adult liver than 4% formaldehyde. The mean penetration rate (±SE) or coefficient of diffusibility of 0.0025% mercaptoethanol-4% formaldehyde into adult liver was 1.32±0.01 and that of 4% formaldehyde was 1.12±0.06 (p<0.04). Both fixatives diffused more rapidly into mouse liver than into human liver. The cross-linking ability of mercaptoethanol-formaldehyde depends on the concentration of the fixative and the time of fixation. Bovine serum albumin (15%) and 0.1% mercaptoethanol alone formed a gel, whilst electrophoresis showed monomers in the supernatant. Mercaptoethanol (0.1%) also rapidly decreased the absorption at 420 nm, suggesting denaturation. It seems that mercaptoethanol increases the number of thiol groups available to form cross-links with formaldehyde. This study demonstrated that mercaptoethanol-formaldehyde fixed and cross-linked tissues better than formaldehyde at 3 h and 4 h, but not at 1 h and 2 h. The most effective concentration of mercaptoethanol for tissue fixation in 4% formaldehyde is 0.0025%.     

Complete Staining of Neuromuscular Innervation with Bromoindigo and Silver

Frozen sections of musde fixed in buffered formaldehyde (pH 7.3) are first incubated for localization of esterase using 5-bromoindoxyl acetate as substrate. The sections are then mounted on slides and stained by a urea-silver nitrate method for axons. Result: subneural apparatus—blue; axons—black; other tissue components in various shades of grey.     

混合甲醛固定液固定大肠癌淋巴结标本的最佳免疫组化效果研究

目的:探讨混合甲醛固定液固定大肠癌淋巴结标本的最佳免疫组化效果。方法:采用不同pH值(6.0、7.0、8.0)的混合甲醛固定液对39枚大肠癌淋巴结标本进行不同时间(6 h、6 h-12 h、1 d-7 d)的固定处理。以细胞角蛋白20(CK20)为目标抗原,运用OIympusdp 70图像采集分析仪抽选出混合甲醛固定液最佳免疫组化染色的pH值及固定时间。结果:经pH值为7.0混合甲醛固定液处理后,阳性率为92.31%,高于经pH值为6.0、8.0的混合甲醛固定液处理后的76.92%、74.36%,且经pH值为7.0、8.0处理后的阳性率比较有统计差异(P0.05)。混合甲醛固定液的固定时间在6 h-12 h时的阳性率为94.87%,高于固定时间为6 h、1 d-7 d处理的30.77%、76.92%(P0.05)。结论:对于大肠癌淋巴结标本,以CK20为目标抗原,选择pH值为7.0的混合甲醛固定液固定6 h-12 h能够得到质量较佳的免疫组化染色效果。     

The Role of DNase and EDTA on DNA Degradation in Formaldehyde Fixed Tissues

Degradation and extraction of high molecular weight DNA from formaldehyde fixed tissues suitable for gene analysis are presented. We previously reported that DNase might play an important role in the degradation of DNA extracted from formaldehyde fixed tissues (Tokuda et al. 1990). In the present study, DNase activity of the supernatant from rat tissues fixed in buffered formaldehyde at room temperature was negligible within 3 hr. Analysis of DNA extracted from reconstituted chromatin revealed that the degradation increased in the absence of DNase depending on the duration of the formaldehyde fixation. Furthermore, high molecular weight DNA could be extracted from tissues devoid of DNase activity fixed in buffered formaldehyde containing EDTA. These results demonstrated that DNA degradation was due mainly to a mechanism other than DNAse which was inhibited by EDTA. For clinical application, v-H-ras gene was successfully detected by Southern blotting from rat spleen tissues fixed in buffered formaldehyde especially at 4 C. Fixation at low temperature is useful for gene analysis.     

The influence of formaldehyde fixation time on the rate of nitrous acid deamination of erythrocytes and spinal cord proteins

Summary Alcohol fixed blood films and fresh blocks of spinal cord were immersed in phosphate buffered neutral 10% formol for graded intervals, the films for 10, 30 min, 1, 2, 4, 8, 24 hr; the blocks for 2, 4, 6, 24 hr at 3 and 24° C; 1, 3, 7, 14, 21, 28, 42, 56 da, 3 and 14 mo at 24–26°. Graded deaminations in 2 N NaNO₂/HAc at 3° C were applied: 1, 2, 5, 10, 20, 30 min; 1, 2, 4, 6, 8, 12, 18, 24, 36 hr. Blood films were stained at pH 6 and 6.5, tissue at pH 4.5 and 5.0, both in azure A eosin B. The point at which erythrocytes reached a slightly bluish green was taken as the end point, since no further color change occurred on further exposure and erythrocytes were the last of usually deamination susceptible tissue elements to lose their oxyphilia on deamination. Deamination of alcohol fixed blood films is completed in about 2 min, of sublimate fixed spinal cord in about 1 hr. Progressive formaldehyde exposure increased deamination time of blood films to 10–20 min in 1 hr, to 6–8 hr in 4 hr and to 12 hr in 24 hr. The tissue deamination showed similar progressive increase of deamination time, slower with 3° C fixation than with 24–26°, reaching 18–36 hr by about 3 days formol, and remaining about the same thereafter.Supported by National Cancer Institute Grant No. C-04816, National Institutes of Health.     

Kinetic Studies of Formaldehyde Binding in Tissue

Specimens of rabbit liver were fixed for various periods up to 6 days in buffered ¹⁴C-formaldehyde. Binding of the isotope reached a plateau after fixation for approximately 24 hr; the half-maximal binding level was reached after approximately 100 min. Formaldehyde binding at 37 C was faster than at 25 C, and faster at pH 7.0 than at pH 4.0. During rinsing of the fixed tissue in water for up to 26 days there was a progressive decrease in isotope content to 10-20% of the pre-rinse level, indicating that formaldehyde fixation is a reversible process.     

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.     

Spermatogenesis in animals as revealed by electron microscopy

Summary Testes of the Japanese crayfish, Cambaroides japonicus, were fixed in buffered (pH 7.4) 4% formaldehyde followed by buffered (pH 7.4) 1% osmium tetroxide, and thin sections of the epoxy Epon resin-embedded tissue were studied with the electron microscope. Spermatozoa from vasa deferentia and spermatids from the testis were examined in smear preparations and thick sections by an ordinary light microscope, employing the Feulgen nuclear technique, fast green or periodic acid-Schiff reagent. On the other hand, testes fixed with buffered (pH 7.4) 4% formaldehyde were incubated in Novikoff and Goldfischer's medium or in Mölbert and coworkers' mixture for demonstrating thiamine pyrophosphatase (TPPase) or alkaline phosphatase, and observed in the electron microscope.The microtubules 220 Å to 310 Å in diameter appearing in the nuclear process seem to represent some unit structure of chromosomes in this species. The microtubules are composed of the tubular subunits which are disposed twisted along the peripheral part of major axis of the microtubules. Such tubular subunits are approximately 20 Å thick in wall and 10 Å wide in lumen. The acrosome in a helmet-like shape has been found to have a hornlike process at its proximal part, though the function of such process remains unsettled in the present study. With incubation in disodiumphenylphosphate, no final product is deposited in any part of the premature spermatozoa. The convoluted membrane as well as the invagination of nuclear envelope are revealed to be specific sites for TPPase activity, and such finding suggests that TPPase may act as an intermediary in formation of nuclear processes of the crayfish sperm.This study was supported by Grant GM-8327-05 from the United States Public Health Service.Scientist from the Laboratory of Electron Microscopy, Department of Biology, Kyung Pook National University, Taegu, Korea.     

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.     

Spermatogenesis in animals as revealed by electron microscopy

Summary Testes of the pond snail, Cipangopaludina malleata Reeve, were fixed in 1% osmium tetroxide, 3% permanganate, or 4% formaldehyde followed by 1% osmium tetroxide, each being buffered to pH 7.2 with Veronal-acetate or Sörensen's phosphate buffer. On the other hand, testes fixed with 4% formaldehyde adjusted to pH 7.2 with 0.075 M Na-cacodylate were incubated in Novikoff-Goldfischer medium for demonstrating thiamine pyrophosphatase, uridine or inosine diphosphatase, uridine monophosphatase or adenosine triphosphatase. The specimens incubated were postfixed in 1% osmium tetroxide buffered to pH 7.2 with Veronal-acetate buffer. Thin sections of the epoxy Epon resin-embedded tissue were stained either singly with saturated aqueous uranyl acetate or doubly with saturated aqueous uranyl acetate followed by lead citrate.In a concentric lamellar structure consisting of the granular endoplasmic reticulum in the cytoplasm of early atypical spermatids, disappearance of ribosomes attached to the outer surface of cisternae seems to have initiated at the central part of the structure, and the cisterna-attached ribosomes seem to participate in the formation of dense granules appearing in the vesicles representing the endoplasmic reticulum of atypical spermatids.The Golgi apparatus of the atypical spermatids in the advanced stages of development is composed of at least three different layers, the central part consisting of an amorphous material, the following lamellar and vesicular elements, and the peripheral fine vesicles.It has been assumed that the mechanism by which the nucleic acid, especially DNA is converted into the polysaccharide might be attributed to the function of the Golgi apparatus, because the transformation of dense granules into less dense granules as well as diphosphatase activities have been detected within the Golgi apparatus.This study was supported by Grant GM-8327-06 from the United States Public Health Service.     

Genetic engineering of stimuli-sensitive silkelastin-like protein block copolymers

Differentially charged analogues of block copolymers containing repeating sequences from silk (GAGAGS) and elastin (GVGVP) were synthesized using genetic engineering techniques by replacing a valine residue with glutamic acid. The sensitivity to pH and temperature was examined at various polymer concentrations, ionic strengths, and polymer lengths. The polymers transitioned from soluble to precipitate state over narrow temperature ranges. The transition temperature T(t) (the temperature at which half-maximal spectrophotometric absorption was observed) increased with increasing pH up to pH 7.0 and leveled off above this value for the Glu-containing polymer (17E)(11). T(t) was independent of pH for the Val-containing polymer (17V)(11). It decreased with increasing ionic strength, polymer concentration, and polymer length for both polymers. These results suggest that by substituting charged amino acids for neutral amino acids at strategic locations in the polymer backbone and by control of the length of silkelastin-like block copolymers using genetic engineering techniques, it is possible to precisely control sensitivity to pH, temperature, and ionic strength.     

The influence of metal cations and pH on the heat sensitivity of photosynthetic oxygen evolution and chlorophyll fluorescence in spinach chloroplasts

The heat-sensitivity of photosynthetic oxygen evolution of thylakoids isolated from spinach increases by increasing the pH above neutral value. The temperature for inactivation (transition temperature) is lowered from about 45° C (pH 6.0–7.4) to 33°C (pH 8.5). Similar results are obtained with intact chloroplasts. At pH 7.0 the transition temperature of washed thylakoids decreases by lowering the salt concentration below 20 mM with monovalent cations (Li⁺, Na⁺, K⁺) and below 3–4 mM with divalent cations (Mg²⁺, Ca²⁺, Sr²⁺). Illumination decreases the heat-sensitivity of oxygen evolution in intact chloroplasts, but even increases the heat-sensitivity in uncoupled chloroplasts. In intact chloroplasts the transition temperature of the heat-induced rise in chlorophyll fluorescence yield (F_o; see Schreiber and Armond 1978) decreases from 44° C to 38° C when the pH of the suspending medium is increased from 6.5 to 8.5. At 20° C, F_o is almost insensitive to pH (6.0–8.5). At 40° C, however, F_o is constant between 6.0 and 7.0, but strongly increases by increasing the pH above neutral value. The results are discussed in terms of a close relation between electrostatic forces at the thylakoid membrane and thermal sensitivity of photosynthetic apparatus. It is suggested that the heat-sensitivity of the photosystem II complex partially depends on the ionization state of fixed groups having alkaline pK. The packed volume of thylakoids suspended in a low salt medium increases when the temperature is increased above 30° C (pH 7.0) and above 20° C (pH 8.0), respectively. This result suggests a heat-induced increase in surface charge density of the thylakoid membrane.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MES morpholinoethane sulfonic acid - MOPS 2-N-morpholinopropane sulfonic acid - TRICIN N-[tris(hydroxymethyl)-methyl] glycine     

Visualization of Legionella Pneumophila in Paraffin Sections using Hexamine Silver

A modification of Gomori's hexamine silver technique is given as a simple, reliable method for the nonspecific demonstration of Legionella pneumophila in paraffin sections. When tested against serogroups I to VI it was found that pretreatment with potassium dichromate rendered L. pneumophila demonstrable by the Gomori-Burtner hexamine silver solution when buffered to pH 7.8. Tissue was fixed in 10% buffered formalin and sections were cut at 3-5 μm. After treatment with 10% potassium dichromate for 1 hour at room temperature, sections are placed in the silver solution at 56 C until they develop a pale golden yellow color, at which point they are checked periodically under the microscope for optimal staining (approximately 3-4 hours). Sections are then toned, fixed and counterstained in 1% neutral red. The L. pneumophila coccobacilli stain black against a clear background, while nuclei stain red/black.     

Peroxidase Oxidation of Phenols

Partially purified preparations of horseradish peroxidase were able to catalyze the effective transformation of such phenol compounds as phenol, o-chlorophenol, 2,4,6-trichlorophenol, pentachlorophenol (giving rise to the formation of polymer products insoluble in water), resorcinol, and thymol (giving rise to the formation of low-molecular-weight products). The following conditions were found to be optimal for peroxidase oxidation and provide the maximum extent of elimination of phenol compounds: temperature, 15–25 and 25–30°C for phenol and chlorophenol compounds, respectively; molar ratio H₂O₂/phenol, 1 : 1; and transformation time, 1–3 h. Although effective transformation was observed within a broad range of pH, the efficiency of the process slightly increased at a pH from 6.0 to 7.5. It was suggested to carry out multiple peroxidase oxidations of phenols using partially purified peroxidase enclosed in a dialysis membrane bag placed into a solution of a phenol compound containing hydrogen peroxide.     

Immunofluorescence Microscopy of Cyanurated Tissues

Test tissues consisted of: (1) popliteal lymph nodes of rabbits, removed 6 hr after injection of hind footpads with 0.2 ml of 125 mg/ml solution of 5× crystallized chicken ovalbumin, and (2) lungs from guinea pigs, passively sensitized with rabbit antiovalbumin serum, then anaphylactically shocked by intracardial injection of a 1% chicken ovalbumin solution. Similar control tissues from normal rabbits, and lungs of passively sensitized guinea pigs, but shocked with histamine instead of ovalbumin, were included. Pieces of fresh tissue not exceeding 2 mm³ were fixed as follows: (1) Cyanuration—lymph nodes, 1 hr; lung, 0.5 hr; both at 23-27 C—in anhydrous methanol containing 0.5% w/v cyanuric chloride and 1% v/v N, N-diethylaminoethanol. (2) Control fixatives—all specimens 18-24 hr at 4—6 C—absolute methanol; 95% ethanol; neutral buffered 10% formalin; and an FAA mixture (formalin, conc., 6; glacial acetic acid, 2; 30% ethanol, 92). Freeze-dried material was either left unfixed (a control) or fixed in xylene or toluene containing 0.5% w/'v cyanuric chloride and 1% v/v N, N-diisopropylaminoethanol; time and temperature as for fresh tissues. All tissues were routinely dehydrated, cleared, and vacuum embedded in an ester wax, diethylene glycol distearate, or in paraffin at 52 C. Sections 2-4 μ thick were attached to gelatin-coated slides, the wax removed in petroleum ether, and stained 20 min at 23-27 C in a 0.10% solution of fluorescein isothiocyanate-conjugated rabbit antiovalbumin globulin, washed in phosphate buffered saline 10 min, dehydrated, cleared and covered in a nonfluorescent medium. With ultraviolet illumination, brightly immunofluorescent, anti-genically specific staining was obtained in cyanurated fresh and freeze-dried lymph node and lung tissues. In contrast, specific staining was diminished or absent in comparable tissues reacted in the control fixatives.     

Ultrastructural visualization of elastic fibres with a tannate-metal salt method

Summary A modification of the tannic acid-metal salt method was applied as an ultrastructural stain for elastin. Thin sections of glutaraldehyde-fixed, embedded rat aorta and rabbit elastic cartilage, with and without osmication, were examined. Raising the pH of the tannic acid solution from 2.7 to 9.0 progressively increased the electron-density of elastic fibres and collagen fibrils in osmicated and unosmicated specimens. The maximum tannic acid staining of elastic fibres was observed in the pH range 7.0–9.0. Collagen staining, although less intense than that of elastic fibres, was also greatest in this pH range. Elastic fibres in osmicated specimens demonstrated the strongest tannic acid staining with a minimal increase in density of collagen and cell nuclei when compared to the unosmicated specimens. Sequential treatments of osmicated specimens with tannic acid pH 7.0–9.0, and uranyl acetate, pH 4.1, enhanced the density of the elastin intensely, increased collagen staining moderately, but hardly increased the density of nuclei and microfibrils. In elastase-digested osmicated specimens, all tannic acid (pH 7.0)-uranyl acetate-reactive elastin was selectively removed. These results demonstrate that all the neutral and alkaline tannic acid-uranyl acetate methods can be used as a postembedment stain for elastin specimens fixed in glutaraldehyde and osmium tetroxide.