Determination of optimal rehydration,fixation and staining methods for histological and immunohistochemical analysis of mummified soft tissues

During an excavation headed by the German Institute for Archaeology, Cairo, at the tombs of the nobles in Thebes-West, Upper Egypt, three types of tissues from different mummies were sampled to compare 13 well known rehydration methods for mummified tissue with three newly developed methods. Furthermore, three fixatives were tested with each of the rehydration fluids. Meniscus (fibrocartilage), skin, and a placenta were used for this study. The rehydration and fixation procedures were uniform for all methods. The stains used were standard hematoxylin and eosin, elastica van Gieson, periodic acid-Schiff, and Grocott, and five commercially obtained immunohistochemical stains including pancytokeratin, vimentin, alpha-smooth-muscle-actin, basement membrane collagen type IV, and S-100 protein. The sections were examined by transmitted light microscopy. Our study showed that preservation of the tissue is dependent on the quality and effectiveness of the combination of the rehydration and fixation solutions, and that the quality of the histological and histochemical stains is dependent on the tissue quality. In addition, preservation of the antigens in the tissues is dependent on tissue quality, and fungal permeation had no influence on the tissue. Finally, the results are tissue specific. For placenta the best solution combination was Sandison and solution III (both fixed with formaldehyde) while results for skin were best with Ruffer I (using formaldehyde and Schaffer as fixatives), Grupe et al. (using formaldehyde as a fixative) and solution III (in combination with formaldehyde and Bouin fixatives). Ruffer II (using formaldehyde as a fixative) and solution III (in combination with Schaffer fixative) gave the best results for fibrocartilage.     

Determination of optimal rehydration, fixation and staining methods for histological and immunohistochemical analysis of mummified soft tissues.

During an excavation headed by the German Institute for Archaeology, Cairo, at the tombs of the nobles in Thebes-West, Upper Egypt, three types of tissues from different mummies were sampled to compare 13 well known rehydration methods for mummified tissue with three newly developed methods. Furthermore, three fixatives were tested with each of the rehydration fluids. Meniscus (fibrocartilage), skin, and a placenta were used for this study. The rehydration and fixation procedures were uniform for all methods. The stains used were standard hematoxylin and eosin, elastica van Gieson, periodic acid-Schiff, and Grocott, and five commercially obtained immunohistochemical stains including pancytokeratin, vimentin, alpha-smooth-muscle-actin, basement membrane collagen type IV, and S-100 protein. The sections were examined by transmitted light microscopy. Our study showed that preservation of the tissue is dependent on the quality and effectiveness of the combination of the rehydration and fixation solutions, and that the quality of the histological and histochemical stains is dependent on the tissue quality. In addition, preservation of the antigens in the tissues is dependent on tissue quality, and fungal permeation had no influence on the tissue. Finally, the results are tissue specific. For placenta the best solution combination was Sandison and solution III (both fixed with formaldehyde) while results for skin were best with Ruffer I (using formaldehyde and Schaffer as fixatives), Grupe et al. (using formaldehyde as a fixative) and solution III (in combination with formaldehyde and Bouin fixatives). Ruffer II (using formaldehyde as a fixative) and solution III (in combination with Schaffer fixative) gave the best results for fibrocartilage.     

The titration of tetanus antitoxin V. Effect of formalization method for the fixation of sheep erythrocytes on the indirect haemagglutination test

Two methods of fixation of sheep erythrocytes with formaldehyde for the titration of tetanus antitoxin by the indirect haemagglutination (IHA) test have been compared. The cells fixed with 3% formaldehyde at 4-8 degrees C for 24 h (formaldehyde (I) fixed cells) were less sensitive than the cells fixed with 3% formaldehyde at 4-8 degrees C for 24 h and subsequently treated with 40% formaldehyde at 4-8 degrees C for a further 24 h (formaldehyde (II) fixed cells). The correlation between the toxin neutralization (TN) and IHA titres using formaldehyde (I) fixed cells was better than that obtained with formaldehyde (II) fixed cells. There was no statistically significant difference between TN and IHA titres after treatment of the sera with 2-Mercaptoethanol using formaldehyde (I) fixed cells. Formaldehyde (I) fixed cells can be used for two months with adequate sensitivity to detect the minimum protective level of tetanus antitoxin in the sera.     

A fine structural study of microwave fixation of tissues

Microwave fixed liver and kidney tissues were examined by electron microscopy. It was found that the preservation of fine structure of these tissues by this method is equal to that processed by routine methods. No difficulty was encountered in sectioning microwave fixed tissue blocks. It is obvious that microwave fixation is a faster and more efficient method.     

A comparison of microwave heating and proteolytic pretreatment antigen retrieval techniques in formalin fixed, paraffin embedded tissues

Antigen retrieval (AR) is a technique that re-exposes epitopes in formalin fixed, paraffin embedded sections and makes them detectable by immunohistochemistry. We compared the effects of two AR procedures, enzyme digestion and microwave heating, on immunostaining of vimentin and desmin in formalin fixed, paraffin embedded tissues. Our results showed that AR is necessary for vimentin and desmin immunostaining in tissues fixed in formalin for more than 48 h. With prolonged fixation times, microwave heating showed better results than enzyme digestion for AR. The same results were obtained using 1% zinc sulfate or Citra Plus solution as retrieval solutions for microwave heating. We recommend microwave heating for AR, because it is easier to use and produces better results compared to enzyme treatment.     

A comparison of microwave heating and proteolytic pretreatment antigen retrieval techniques in formalin fixed,paraffin embedded tissues

Antigen retrieval (AR) is a technique that re-exposes epitopes in formalin fixed, paraffin embedded sections and makes them detectable by immunohistochemistry. We compared the effects of two AR procedures, enzyme digestion and microwave heating, on immunostaining of vimentin and desmin in formalin fixed, paraffin embedded tissues. Our results showed that AR is necessary for vimentin and desmin immunostaining in tissues fixed in formalin for more than 48 h. With prolonged fixation times, microwave heating showed better results than enzyme digestion for AR. The same results were obtained using 1% zinc sulfate or Citra Plus solution as retrieval solutions for microwave heating. We recommend microwave heating for AR, because it is easier to use and produces better results compared to enzyme treatment.     

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%.     

Microwave irradiation for fixation and immunostaining of endothelial cells in situ

Using microwave irradiation during tissue fixation and immunostaining reduces sample preparation time and facilitates penetration of fixatives and antibody solutions into the tissues. This results in improved fixation and reduction of non-specific binding of antibodies, respectively. Experimental analyses of endothelial cells in blood vessels in situ have been limited because of the difficulty of tissue preparation. We report here a technique using intermittent microwave irradiation for blood vessel fixation and immunostaining the fixed tissues. Intermittent microwave irradiation during fixation reduced blood vessel contraction and resulted in well preserved morphology of blood vessels, especially the endothelial cells. Microwave irradiation also reduced non-specific binding of fluorescein-labeled antibodies. These microwave irradiation-assisted techniques are useful for analysis of endothelial cell function and for pathological study of blood vessels in situ.     

Understanding microwave-stimulated Romanowsky-Giemsa staining of plastic embedded bone marrow

Summary Bone marrow smears were made and fixed in methanol or formaldehyde. Marrow sections of various thicknesses were also prepared from formaldehyde fixed marrows embedded in paraffin or plastic (glycol methacrylate). The different smears and sections were then stained by a Romanowsky-Giemsa procedure. Some specimens were stained using a standard microwave-stimulated method previously used diagnostically. The effects of technical variations were studied, including degree of microwave irradiation and the staining time. Comparisons of the resulting staining outcomes showed that microwave stimulated Romanowsky-Giemsa staining of plastic sections is a rate controlled process. Unusual aspects of the staining pattern of plastic sections (namely the purple basophilic cytoplasms and nucleoli, and blue chromatin) are due to microwave stimulation and formaldehyde fixation respectively.     

The Use of Mouse Models of Breast Cancer and Quantitative Image Analysis to Evaluate Hormone Receptor Antigenicity after Microwave-assisted Formalin Fixation

Microwave methods of fixation can dramatically shorten fixation times while preserving tissue structure; however, it remains unclear if adequate tissue antigenicity is preserved. To assess and validate antigenicity, robust quantitative methods and animal disease models are needed. We used two mouse mammary models of human breast cancer to evaluate microwave-assisted and standard 24-hr formalin fixation. The mouse models expressed four antigens prognostic for breast cancer outcome: estrogen receptor, progesterone receptor, Ki67, and human epidermal growth factor receptor 2. Using pathologist evaluation and novel methods of quantitative image analysis, we measured and compared the quality of antigen preservation, percentage of positive cells, and line plots of cell intensity. Visual evaluations by pathologists established that the amounts and patterns of staining were similar in tissues fixed by the different methods. The results of the quantitative image analysis provided a fine-grained evaluation, demonstrating that tissue antigenicity is preserved in tissues fixed using microwave methods. Evaluation of the results demonstrated that a 1-hr, 150-W fixation is better than a 45-min, 150-W fixation followed by a 15-min, 650-W fixation. The results demonstrated that microwave-assisted formalin fixation can standardize fixation times to 1 hr and produce immunohistochemistry that is in every way commensurate with longer conventional fixation methods.     

The use of Histochoice™® for histological examination of articular and growth plate cartilages,intervertebral disc and meniscus

Histochoice?® is a proprietary nontoxic, non-cross-linking fixative designed by the manufacturer to replace formaldehyde based fixation protocols. We compared Histochoice and formalin fixation for several cartilaginous tissues including, articular and growth plate cartilage, meniscus and intervertebral disc. The tissues were stained with general histology stains including toluidine blue for tissue proteoglycans, picrosirius red to evaluate collagenous organization, and hematoxylin and eosin to assess cell morphology. The chondroitin sulfate and heparin sulfate substituted proteoglycans aggrecan and perlecan were also immunolocalized in some of the tissues to provide a comparison. Histochoice did not fix deep into the tissue blocks resulting in focal loss of aggrecan and other matrix components from the more central regions of the blocks. This was evident in toluidine blue stained sections of immature tibial articular cartilage where loss of glycosaminoglycan was significant in Histochoice?® fixed tissues. Histochoice fixation worked well, however, in the aggrecan and perlecan immunohistology applications where its non-cross-linking traits were conducive to epitope retrieval and identification by primary antibodies to extracellular matrix components.     

Tissue Fixation for Immunohistochemical Detection of Proliferating Cell Nuclear Antigen with PC10 Monoclonal Antibody

PC10 is a monoclonal antibody to proliferating cell nuclear antigen, a nuclear protein associated with the cell cycle. We have evaluated the effects of tissue fixation on PC10 immunoreactivity in sections of paraffin embedded rat tissues. Immunoreactivity was well preserved in tissues after fixation with alcohol-based solutions for 3-24 hr. Fewer PC10-positive cells were detectable in samples fixed with formaldehydecontaining solutions compared with samples fixed with alcohol for the same time. Loss of PC10 immunoreactivity in formaldehyde fixed tissues was progressive, and quantifiable as early as after 3 hr fixation. Consequently, alcohol-based fixatives are strongly recommended for any immunocytochemical prospective study using PC10 antibody. In contrast, loss of PC10-immunoreactivity is always predictable, but difficult to quantitate, using formaldehyde fixed specimens. This aspect should be considered when using PC10 antibody in retrospective studies with routinely-processed archival material.     

Cedarwood Oil as a Clearing Agent with Acetic-Alcohol Fixatives

PC10 is a monoclonal antibody to proliferating cell nuclear antigen, a nuclear protein associated with the cell cycle. We have evaluated the effects of tissue fixation on PC10 immunoreactivity in sections of paraffin embedded rat tissues. Immunoreactivity was well preserved in tissues after fixation with alcohol-based solutions for 3–24 hr. Fewer PC10-positive cells were detectable in samples fixed with formaldehydecontaining solutions compared with samples fixed with alcohol for the same time. Loss of PC10 immunoreactivity in formaldehyde fixed tissues was progressive, and quantifiable as early as after 3 hr fixation. Consequently, alcohol-based fixatives are strongly recommended for any immunocytochemical prospective study using PC10 antibody. In contrast, loss of PC10-immunoreactivity is always predictable, but difficult to quantitate, using formaldehyde fixed specimens. This aspect should be considered when using PC10 antibody in retrospective studies with routinely-processed archival material.     

Microwave fixation of the lung

A technique for microwave fixation of inflated rat lung is described. Conventional intratracheal fixation with instillation of fixative into the airways at a constant pressure results in pressure artifacts as well as flushing and disruption of cells and exudates. Microwave fixation fixes these elements in situ without disruption and thus is valuable when evaluating the distribution of inflammatory infiltrates. Exudative pneumonitis was produced in the rat using intratracheal instillations of either endotoxin or silica and comparisons were made between histologic sections fixed using either standard formalin fixation or microwave fixation.     

Microwave-stimulated glutaraldehyde and osmium tetroxide fixation of plant tissue: ultrastructural preservation in seconds.

Microwave-enhanced fixation of animal tissues for electron microscopy has gained in interest in recent years. Attempts to use microwave irradiation for the preparation of plant tissues are rare. In this study; I report on microwave conditions which allow a high quality preservation of plant cell structure. Tissues used were: internodes of Chara vulgaris, leaves of Hordeum vulgare, root tips of Lepidium sativum. Microwave irradiation was done with a commercial microwave oven (Sharp R-5975). Fixatives used were: 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 and 1% osmium tetroxide in veronal/acetate buffer, pH 7.2. Conventional fixations with glutaraldehyde/osmium were compared with microwave fixations. Examinations of thin sections showed that microwave fixation (glutaraldehyde or sequential aldehyde/osmium) is an attractive and rapid alternative method for processing plant tissues for electron microscopy. The optimal conditions found were: microwave oven at power level 50 W, 6.5 ml of fixative solution, irradiation times between 32-34 s, final temperature between 40 degrees C and 47 degrees C.     

Microwave Fixation of the Lung

A technique for microwave fixation of inflated rat lung is described. Conventional intratracheal fixation with instillation of fixative into the airways at a constant pressure results in pressure artifacts as well as flushing and disruption of cells and exudates. Microwave fixation fixes these elements in situ without disruption and thus is valuable when evaluating the distribution of inflammatory infiltrates. Exudative pneumonitis was produced in the rat using intratracheal instillations of either endotoxin or silica and comparisons were made between histologic sections fixed using either standard formalin fixation or microwave fixation.     

Microwave oven antigen retrieval applied to the immunostaining of cytopathology specimens

Microwave oven antigen retrieval has been developed to extend the range of antibodies that can be used upon sections of fixed and processed tissue. It has the additional advantages of improving immunostain intensity and reducing background positivity. It can also be employed as an alternative to proteolytic digestion. In this study the effects of microwave oven heating upon immunochemical staining of cytopathological specimens with a range of selected antibodies have been investigated. Microwaving did not result in loss of cells, and there was no need to use adhesive-coated slides. the method improved the staining intensity and reduced background with antibodies against a variety of antigens that are difficult or impossible to detect in formaldehyde-fixed cytological material. Microwave heating was also used successfully as an alternative to trypsin digestion, and had the advantage of reduced morphological distortion. the technique was useful in demonstrating the soluble formalin-sensitive antigen p19 on cytospins fixed in formaldehyde vapour. Microwave oven heating thus shows promise of extending the scope of immunostaining in clinical cytopathology.     

Effects of antigen retrieval by microwave heating in formalin-fixed tissue sections on a broad panel of antibodies

Formaldehyde fixation of biopsy specimens for routine purposes has often been held responsible for the poor reproducibility of immunohistochemical studies. Recently, antigen retrieval (AGR) using microwave irradiation was described as a potential tool to enhance immunostaining. A comparison of conventional staining and staining after microwave heating was performed for 52 markers, using tissues fixed in formaldehyde for 24 h, 1 to 6 weeks and 3 years respectively, as well as consultant case material. After adequate duration of fixation (24 h), only a few markers (17%) showed better results after AGR, but this percentage was increased to 50% when tissues were fixed for longer periods. Maximal enhancement was obtained in the group of consultant cases (58% of tested markers demonstrated better staining results), in which the period of fixation and tissue processing was unknown. To achieve reliable enhancement with AGR, continuous heating (100° C) should not be shorter than 20 min. In conclusion, AGR may become the most important tool to simplify and equalize immunohistochemical techniques, if critically evaluated.     

Tissue fixation with phenol-formaldehyde for routine histopathology

Summary The addition of 2% phenol had a marked accelerating effect on neutral buffered 4% formaldehyde as a fixative. Histopathological material fixed in buffered phenol—formaldehyde (pH7.0) and rapidly advanced to paraffin in an enclosed tissue-processor showed improved nuclear and cytoplasmic detail, reduced shrinkage and distortion, and an absence of formalin pigment. Good results were obtained in less time when sequential fixation in phenol—formaldehyde buffered to pH7.0 and pH5.5 was carried out at an elevated temperature (40°C) in the enclosed tissue-processor. Standard histological stains and immunoperoxidase methods worked well. In resin-embedded tissue, buffered phenol—formaldehyde (pH7.0) gave satisfactory ultrastructural results. The penetration rate of buffered phenol—formaldehyde (pH7.0) in gelatin models did not differ from that of neutral buffered 4% formaldehyde. Polyacrylamide gel electrophoresis showed enhanced protein polymer formation with buffered phenol—formaldehyde (pH7.0) as compared with neutral buffered 4% formaldehyde. Protein polymer formation increased in response to increased time and temperature. Cells fixed in suspension in buffered phenol—formaldehyde (pH7.0) and neutral buffered 4% formaldehyde showed similar volume changes.     

甲醛固定对GFP发光特性的影响

绿色荧光蛋白(GFP)能够作为报告分子对活体细胞中特定基因的时空表达进行实时追踪,因此广泛应用于生物学研究领域。在用GFP对细胞活动进行追踪的实验中,常有无法在取样后及时对样品进行荧光观察的情况,此时需要先将样品进行固定以便对其进行观测。然而,不恰当的细胞固定方法会导致胞内GFP荧光信号强度减弱、位置改变等后果。甲醛是最常用的细胞固定剂,也常被用于固定表达GFP蛋白的细胞样品。但对甲醛固定GFP样品的报道多是针对于真核细胞,且固定效果也存在较大差异。文章系统地探索了甲醛浓度、固定时间、固定缓冲液种类对两种细菌(E.coli及鱼腥蓝细菌Anabaena PCC7120)胞内GFP信号的影响。结果显示,较低浓度(≤1%)的甲醛处理2h后,细胞的荧光强度在1d后仍可保持80%以上,胞内荧光点无弥散现象发生。具有相近pH的几种常见缓冲液对荧光强度的影响无显著差异。随着甲醛浓度的增加、固定时间的延长、溶液pH的增加(中性至偏碱性),细胞中的荧光强度会逐渐降低。