Fixation,decalcification, and tissue processing effects on articular cartilage proteoglycans

Summary Neutral buffered 4% formaldehyde fixation for 48h preserved well the proteoglycan content of bovine articular cartilage. Neither subsequent demineralization in 10% EDTA, nor light microscopic tissue processing, reduced the hexosamine or uronic acid content of the tissue. Fixation in alcoholic solutions increased Safranin O binding as well as periodic acid Schiff reaction of the cartilage matrix as measured by microspectrophotometry. It is suggested that the enhanced staining of cartilage was due to better preservation of the glycoprotein oligosaccharides. Quaternary ammonium salts in the fixative suppressed the staining of cartilage matrix with Safranin O.     

The effects of different decalcification protocols on TUNEL and general cartilage staining.

Apoptosis is characterized by DNA strand breaks with a 3'-OH terminus, which are analyzed by terminal deoxy(d)-UTP nick end labeling (TUNEL). Proteinase K digestion is thought to be an essential step in the TUNEL procedure. The effects of decalcifying reagents on general staining and the TUNEL assay for cartilage sections are largely unknown. The effects of these reagents on retention and integrity of DNA in chondrocytes have not been described until now. We evaluated the effects of various decalcifying solutions, including 10% EDTA, 10% citric acid, 5% trichloroacetic acid, 5% acetic acid and a commercial hydrochloric acid-based reagent, on general cartilage staining and the TUNEL assay for cartilage. The effects of proteinase K on nucleus preservation were also examined. Decalcification with 10% EDTA gave the best result for general cartilage staining. Chondrocyte DNA was retained and intact after using this reagent. Decalcification with 10% EDTA is also the safest method of decalcification if the TUNEL assay is applied to cartilage. Proteinase K digestion may have adverse effects on nucleus preservation in cartilage. Awareness of these effects is important whenever the TUNEL assay is applied.     

Staining of demineralized cartilage

Summary Safranin O in the orthochromatic form stains articular cartilage proteoglycan quantitatively in histological sections of demineralized cartilage. This was shown by scanning microdensitometry of stained sections of undemineralized and demineralized articular cartilage and by biochemical analysis of ³⁵S labelled cartilage subjected to demineralization. In constrast, Alcian Blue staining is affected by unknown factors other than simply the amount of proteoglycan present. Alcoholic formalin fixes articular cartilage proteoglycan more successfully than formol Zenker for subsequent rapid demineralization. Alcoholic formalin does not preserve cellular appearance as well as formol Zenker. Staining of articular cartilage with PAS appears unaffected by demineralization.     

Alizarin Red S and Toluidine Blue for Differentiating Adult or Embryonic Bone and Cartilage

This technic has been successfully employed by the author for staining, in toto, the bones and cartilage of mature specimens of Urodela and the developing bone and cartilage of the embryonic human, cat, pig and rat. The differential staining is accomplished by using a modification of Dawson's method of staining bone with alizarin red S following a toluidine blue solution specific for cartilage. Specimens are fixed in 10% formalin, stained one week in a solution of .25 g. of toluidine blue in 100 cc. of 70% alcohol, macerated 5 to 7 days in a 2% KOH solution, counterstained for 24 hours in a 0.001% solution of alizarin red S in 2% aqueous KOH, dehydrated in cellosolve and cleared in methyl salicylate. In the adult and embryonic forms thus treated the soft tissues are cleared while the osseous tissue is stained red, the cartilage blue.     

A procedure for differential staining of cartilage and bone in whole formalin-fixed vertebrates.

This paper describes a modification of the Simons and Van Horn (1971) procedure for rendering cartilage blue, bone red, and soft tissue translucent or transparent in whole vertebrate specimens. Alcian blue and alizarin red S are used to stain cartilage and bone respectively. In our procedure formalin is used as a fixative. This is a significant modification because formalin is the common fixative for museum specimens. This clearing and staining procedure is thus readily applicable to comparative studies in anatomy, embryology and systematic zoology.     

Histological Technic for Cerebellar Climbing and Mossy Fibers

In order to, avoid disadvantages attendant upon the use of fresh frozen sections, or of block impregnation with silver, in staining climbing or mossy fibers of the cerebellum, Rio Hortega's double impregnation method for nerve fibers is useful. This consists of prolonged formalin fixation prior to cutting frozen sections (which thereafter are easier to cut) and preliminary treatment with ammoniacal aqueous and alcoholic washes, mordanting in pyridine silver, and treatment with pyridine-silver-carbonate. Following this, sections are handled individually through one of several reduction methods after which they may be directly mounted or gold toned.     

The effects of different decalcification protocols on TUNEL and general cartilage staining

Apoptosis is characterized by DNA strand breaks with a 3'-OH terminus, which are analyzed by terminal deoxy(d)-UTP nick end labeling (TUNEL). Proteinase K digestion is thought to be an essential step in the TUNEL procedure. The effects of decalcifying reagents on general staining and the TUNEL assay for cartilage sections are largely unknown. The effects of these reagents on retention and integrity of DNA in chondrocytes have not been described until now. We evaluated the effects of various decalcifying solutions, including 10% EDTA, 10% citric acid, 5% trichloroacetic acid, 5% acetic acid and a commercial hydrochloric acid-based reagent, on general cartilage staining and the TUNEL assay for cartilage. The effects of proteinase K on nucleus preservation were also examined. Decalcification with 10% EDTA gave the best result for general cartilage staining. Chondrocyte DNA was retained and intact after using this reagent. Decalcification with 10% EDTA is also the safest method of decalcification if the TUNEL assay is applied to cartilage. Proteinase K digestion may have adverse effects on nucleus preservation in cartilage. Awareness of these effects is important whenever the TUNEL assay is applied.     

Fixative evaluation and histologic appearance of embryonic rodent tissue

Histopathologic study of early hamster embryos was carried out after fixation in Zenker's solution, alcoholic formalin, Bouin's fluid, 10% neutral buffered formalin, or 3% glutaraldehyde and staining with hematoxylin and eosin. Fixation in Zenker's fluid followed by postfixation in neutral buffered formalin provided superior preservation of normal embryonic subcellular detail as compared to the other candidate processing techniques.     

Enzyme clearing of alcian blue stained whole small vertebrates for demonstration of cartilage.

Preparation of small vertebrates cleared after alcian blue staining of cartilage is facilitated by trypsin digestion. Specimens are fixed in formation, washed, skinned, and eviscerated. After staining in a solution of alcian blue in acetic acid-alcohol for 24-48 hours, they are transferred to water through graded alcohols. Excess alcian blue is removed over a period of up to three weeks by changes every 2-3 days of 1% trypsin in approximately one-third-saturated sodium borate. Bony tissues may be stained after this in a solution of alizarin red S in 0.5% KOH. Specimens are bleached if necessary and dehydrated through graded KOH-glycerine mixtures for storage in glycerine. Since alcohol treatment in addition to formalin fixation does not affect results with this method, it should be useful to researchers who want to study the cartilage or cartilaginous skeletons in museum specimens, which are routinely fixed in formalin and stored in alcohol.     

Stain Technology: Fixative Evaluation and Histologic Appearance of Embryonic Rodent Tissue

Histopathologic study of early hamster embryos was carried out after fixation in Zenker's solution, alcoholic formalin, Bouin's fluid, 10% neutral buffered formalin, or 3% glutaraldehyde and staining with hematoxylin and eosin. Fixation in Zenker's fluid followed by postfixation in neutral buffered formalin provided superior preservation of normal embryonic subcellular detail as compared to the other candidate processing techniques.     

Staining Paraffin-Embedded Plant Tissues with Acridine Orange

Stem, leaf, and bud tissue of sweet potato, tomato, and pepper were embedded in paraffin, sectioned, mounted, and stained with 0.01, 0.1 and 1% aqueous and 0.1% alcoholic solutions of acridine orange. Temporary and durable mounts were prepared and irradiated under short and long wave ultraviolet light. Intensity and specificity of the fluorescence imparted to tissues were chiefly affected by type of fixative. Best results were obtained with fixatives containing formalin but not acetic acid. Tests on the effect of pH obtained with McIlvaine's buffer between 4.5 and 8.3, and made only with the aqueous stain, showed 6-8 to be optimal. Aqueous staining 1 hr in 0.1% solution, pH 6-8 is recommended for temporary mounts. Durable mounts in a nonfluorescent resin can be made after differentiation in buffer and dehydration in dioxan solutions.     

An Ion-exchange Bone Demineralization Method for Improved Time,Expense, and Tissue Preservation

Here, we describe an ethylenediaminetetraacetic acid (EDTA)-based bone demineralization procedure that uses cation-exchange resin and dialysis tubing. This method does not require solution changes or special equipment, is faster than EDTA alone, is cost-effective, and is environmentally friendly. Like other EDTA-based methods, this procedure yields superior tissue preservation than formic acid demineralization. Greater protein antigenicity using EDTA as opposed to formic acid has been described, but we also find significant improvements in carbohydrate-based histological staining. Histological staining using this method reveals cartilage layers that are not distinguishable with formic acid demineralization. Carbohydrate preservation is relevant to many applications of bone demineralization, including the assessment of osteoarthritis from bone biopsies and the use of demineralized bone powder for tissue culture and surgical implants. The improvements in time, expense, and tissue quality indicate this method is a practical and often superior alternative to formic acid demineralization:     

Fixation-dependent cytoplasmic false-positive staining with an immunoperoxidase method

Fixation-dependent nonspecific staining with the unlabeled immunoperoxidase (PAP) method was studied using paraffin-embedded human spleen sections fixed in various fixatives; the specific primary antiserum was omitted or nonimmunized normal rabbit serum was used. Strong cytoplasmic staining of polymorphonuclear leucocytes and macrophages was found after fixation in acetone, alcoholic formalin (94% alcohol) and absolute ethanol. This staining was mainly produced by the second layer of the PAP method. The most probable explanation of this phenomenon is nonspecific protein-immunoglobulin interaction as a result of alcoholic or acetone fixation of the sections. The present findings point to the importance of controls for each case under study to avoid false-positive interpretations.     

Fixation-dependent cytoplasmic false-positive staining with an immunoperoxidase method

Summary Fixation-dependent nonspecific staining with the unlabeled immunoperoxidase (PAP) method was studied using paraffin-embedded human spleen sections fixed in various fixatives; the specific primary antiserum was omitted or nonimmunized normal rabbit serum was used. Strong cytoplasmic staining of polymorphonuclear leucocytes and macrophages was found after fixation in acetone, alcoholic formalin (94% alcohol) and absolute ethanol. This staining was mainly produced by the second layer of the PAP method. The most probable explanation of this phenomenon is nonspecific protein-immunoglobulin interaction as a result of alcoholic or acetone fixation of the sections. The present findings point to the importance of controls for each case under study to avoid false-positive interpretations.Supported by the Sigrid Jusélius Foundation and Finska Läkaresällskapet     

Honey as a substitute for formalin?

Formalin has long been the standard fixative for clinical routines worldwide. After the Formaldehyde Standard became law in the US in 1987, as a result of increasing concerns about the potential carcinogenicity of formaldehyde, attempts have been made to find safer alternatives. Alcoholic formalin is a useful fixative, because in addition to fixation, dehydration also is begun. For centuries, honey has been known to be an antibacterial agent with the potential to preserve compounds without harmful effects on its users. We compared the effects of honey fixation with other routine fixatives using conventional histochemical and immunohistochemical staining methods. Our results demonstrated that tissues fixed in either honey or alcoholic formalin and 10% neutral buffered formalin (NBF) have similar histomorphology. Honey fixation showed minor histomorphological differences among the various tissues; however, it did not influence affect correct diagnostic conclusions. Our results suggested that honey can be used as a safe alternative to formalin in histopathology.     

Enzyme Clearing of Alcian Blue Stained Whole Small Vertebrates for Demonstration of Cartilage

Preparation of small vertebrates cleared after alcian blue Staining of cartilage is facilitated by trypsin digestion. Specimens are fixed in formalin, washed, skinned, and eviscerated. After staining in a solution of alcian blue in acetic acid-alcohol for 24-48 hours, they are transferred to water through graded alcohols. Excess db blue is removed over a period of up to three weeks by changes every 2-3 days of 1% trypsin in approximately we-third-saturated sodium borate. Bony tissues may be stained after this in a solution of alixarin red S in 0.5% KOH. Specimens rue bleached if necessary and dehydrated through graded KOH-glycerine mixtures for storage in glycerine. Since alcohol treatment in addition to formalin fixation does not affect results with this method, it should be useful to researchers who want to study the cartilage or cartilaginous skeletons in museum specimens, which me routinely fixed in formalin and stored in alcohol.     

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.     

Dichromate-Chlorate Perfusion Prior to Staining Degeneration in Brain and Spinal Cord

A method of fixation compatible with both the Nauta-Gygax and Swank-Davenport procedures for degenerating nerve fibers, which shortens the time required by the former procedure, is as follows: The central nervous system is perfused with a 0.9% aqueous solution of NaCl followed by an aqueous solution containing 5% K₂Cr₂O₇ and 2.5% KClO₃. The central nervous system is then hardened in 10% formalin for 1-3 days. Tissue for Marchi-type staining can be taken at this stage. For silver staining, the processing is continued by immersion overnight in 10% formalin in 20% alcohol, and frozen sections cut the next day. Sections, up to 50μ in thickness, are collected in 10% formalin and impregnated by the Nauta-Gygax technique. Best results are obtained by impregnating within 24-48 hr after sectioning.     

Forming and removing stain precipitates on ultrathin sections

Stain precipitates resulting from the use of lead or uranyl salts, or both, on ultrathin sections can be classified as belonging to one of three morphological types: I) extremely electron-dense particles caused by prolonged use of lead salts only, II) amorphous networks formed following double staining with either aqueous or alcoholic uranyl and lead salts, and III) crystalline needles sometimes resulting from double staining with alcoholic uranyl and lead salts. It has been found, however, that either acetic acid or aqueous uranyl acetate can be used to remove type I and type II precipitates from sections, and that oxalic acid and alcoholic uranyl solution will remove type II precipitates. Unfortunately, type III precipitates are unaffected by any agents tested so far.     

Improved Procedure for Histological Identification of Osteoid Matrix in Decalcified Bone

Several improvements on the original method of Yoshiki and coworkers for histological identification of osteoid matrix in decalcified bone are described in this report. The first, fixation of bone with neutral buffered formalin, a popular and stable fixative, should produce better tissue morphology and ensure easy handling in any laboratory. The second is a simple test for aged cyanuric chloride. Aged reagents show poor or no solubility in methanol and have almost no effect on differential staining of osteoid matrix. The third is an application of an organic acid solution in place of neutral EDTA for bone decalcification. Reduced decalcification time with the acid results in rapid preparation of bone sections. Neutral formalin fixation, immersion in the cyanuric chloride solution, decalcification with an organic acid, and hematoxylin and eosin staining, all quite routine laboratory procedures, yield high quality results for identification of osteoid matrix in bone sections.