Improved microradiographic contrast for bone stain-historadiography.

Microradiographs of 5-micron sections of methyl methacrylate embedded undemineralized bone show poor resolution, but prestaining with silver nitrate increases the radioopacity of the calcified tissues to soft x-rays without masking regional differences in microscopic mineralization. Identical results are achieved using aqueous (pH 5.8 and 7.5) or ammoniacal solutions (pH 7.9). Atomic absorption spectrometry detected no loss of calcium from the sections during staining. Osteoid in plastic-embedded bone is not rendered radiopaque by this technique even after prolonged staining times (5 min-2 hr).     

Osteopontin is histochemically detected by the AgNOR acid-silver staining

Silver nitrate staining of decalcified bone sections is known to reveal osteocyte canaliculi and cement lines. Nucleolar Organising Regions (NOR) are part of the nucleolus, containing argyrophilic proteins (nucleoclin/C23, nucleophosmin/B23) that can be identified by silver staining at low pH. The aim of this study was to clarify the mechanism explaining why AgNOR staining also reveals osteocyte canaliculi. Human bone and kidney sections were processed for silver staining at light and electron microscopy with a modified method used to identify AgNOR. Sections were processed in parallel for immunohistochemistry with an antibody direct against osteopontin. Protein extraction was done in the renal cortex and decalcified bone and the proteins were separated by western blotting. Purified hOPN was also used as a control. Proteins were electro-transferred on polyvinylidene difluoride membranes and stained for AgNOR proteins. In bone, Ag staining identified AgNOR in cell nuclei, as well as in osteocyte canaliculi, cement and resting lines. In the distal convoluted tubules of the kidney, silver deposits were also observed in cytoplasmic granules on the apical side of the cells. Immunolocalization of osteopontin closely matched with all these locations in bone and kidney. Ag staining of membranes at low pH revealed bands for NOR proteins and 56 KDa (kidney), 60KDa (purified hOPN) and 75 KDa (bone) bands that corresponded to osteopontin. NOR proteins and osteopontin are proteins containing aspartic acid rich regions that can bind Ag. Staining protocols using silver nitrate at low pH can identify these proteins on histological sections or membranes.     

A simple method using alizarin red S for the detection of calcium in epoxy resin embedded tissue

This report presents a simple procedure for staining 1-2 microns epoxy plastic sections of cells and mineralizing matrix present in fetal bovine bone tissue cultures. A 0.3% aqueous toluidine blue O solution was used as a cellular stain and was followed with 2% alizarin red S for the detection of calcium at sites of mineralization. Effects of concentration and pH of alizarin red S on the penetration of epon embedded thick sections were investigated. Optimal staining was achieved with a 2% aqueous alizarin red S solution adjusted to a pH of 5.5-6.5. This staining procedure provides unusually clear contrast between mineral and bone cells in plastic sections for light microscopy.     

A Simple Method Using Alizarin Red S For the Detection of Calcium in Epoxy Resin Embedded Tissue

This report presents a simple procedure for staining 1-2 μm epoxy plastic sections of cells and mineralizing matrix present in fetal bovine bone tissue cultures. A 0.3% aqueous toluidine blue 0 solution was used as a cellular stain and was followed with 2% alizarin red S for the detection of calcium at sites of mineralition. Effects of concentration and pH of alizarin red S on the penetration of epon embedded thick sections were investigated Optimal staining was achieved with a 2% aqueous alizarin red S solution adjusted to a pH of 5.5-6.5. This staining procedure provides unusually clear contrast between mineral and bone cells in plastic sections for light microscopy.     

Staining and Histomorphometry of Microcracks in the Human Femoral Head

We developed staining techniques that permit identification and histomorphometric analysis of microcracks in the human femoral head 1) from thick, ground bone sections (100 μm) by prestaining with the Villanueva mineralized bone stain (MIBS), and 2) from plastic embedded, undecalcified thin bone sections (5-15 μm) by staining in gallocyanin chrome alum-Villanueva blood stain methods. Both methods represent a significant improvement in the stainability of the microcracks, cellular and tissue elements, and the simultaneous assessment of osteoid seams and tetracycline markers by histomorphometry. Shrinkage and other artifacts were minimized, which helped to clarify some of the uncertainties arising from artifacts resulting from some bone staining methods. Histomorphometric analyses of microcracks were conducted on thick, ground sections of subchondral and trabecular bone. Microcracks were more prevalent in the subchondral bone and osteochondral junction than in the more distant trabeculae. We have consistently localized microcrack areas in bone tissues prepared in these ways.     

Inhibition Reactivation Myofibrillar ATPase Technique for Demonstration of Three Fiber Types in a Single Cryostat Muscle Section

An inhibition reactivation technique was used for histochemical staining of human skeletal muscle sections. Myofibrillar ATPase activity was inhibited by sodium hydroxymercuribenzoate (2.5 mM in 0.1 M Tris-HCl buffer, pH 7.2-7.5, 30 min) and successively reactivated by cysteine which was added to incubation solution (10 mM cysteine-HCl, 2.5 mM ATP-disodium salt, 50 mM potassium chloride and 27 mM calcium chloride in barbital buffer, pH 9.4, 35 min at 37 C). This technique allows the distinction of three fiber categories with different staining intensities in single cross-section. Dark, intermediate and light fibers correspond to IIB, I, and IIA types, respectively. Storage of air dried sections in the freezer at -20 C for one month had no influence on staining characteristics.     

Inhibition reactivation myofibrillar ATPase technique for demonstration of three fiber types in a single cryostat muscle section

An inhibition reactivation technique was used for histochemical staining of human skeletal muscle sections. Myofibrillar ATPase activity was inhibited by sodium hydroxymercuribenzoate (2.5 mM in 0.1 M Tris-HCl buffer, pH 7.2-7.5, 30 min) and successively reactivated by cysteine which was added to incubation solution (10 mM cysteine-HCl, 2.5 mM ATP-disodium salt, 50 mM potassium chloride and 27 mM calcium chloride in barbital buffer, pH 9.4, 35 min at 37 C). This technique allows the distinction of three fiber categories with different staining intensities in single cross-section. Dark, intermediate and light fibers correspond to IIB, I, and IIA types, respectively. Storage of air dried sections in the freezer at -20 C for one month had no influence on staining characteristics.     

Investigation of Thiazin Dyes as Biological Stains II. Influence of Buffered Solutions on Staining Properties

The effect of buffer solutions of varying reaction upon staining fixed sections with thionin, azures A, B, and C, and methylene blue has been studied. The buffer solutions were employed in one of three different ways: for pre-treatment of the sections, for post-treatment, or as solvents for the dyes. Regardless of the method of employing the buffer solutions it was found that the intensity of staining increased with increasing pH-values (a fact which is generally known to be true in the case of basic dyes). It is not certain whether this effect is due to varying the H-ion concentration or to altering the salt content of the solution, or to both. It was also noticed that there was one point where the staining intensify increased most rapidly. This point was either between pH 5 and pH 6 or between pH 6 and pH 7, its position varying with the method of fixation and of applying the buffer solutions. It was further observed that between pH 5 and pH 7 there were always more pronounced metachromatic effects than with either more acid or more alkaline buffer solutions.     

Investigation of Thiazin Dyes as Biological Stains II. Influence of Buffered Solutions on Staining Properties

The effect of buffer solutions of varying reaction upon staining fixed sections with thionin, azures A, B, and C, and methylene blue has been studied. The buffer solutions were employed in one of three different ways: for pre-treatment of the sections, for post-treatment, or as solvents for the dyes. Regardless of the method of employing the buffer solutions it was found that the intensity of staining increased with increasing pH-values (a fact which is generally known to be true in the case of basic dyes). It is not certain whether this effect is due to varying the H-ion concentration or to altering the salt content of the solution, or to both. It was also noticed that there was one point where the staining intensify increased most rapidly. This point was either between pH 5 and pH 6 or between pH 6 and pH 7, its position varying with the method of fixation and of applying the buffer solutions. It was further observed that between pH 5 and pH 7 there were always more pronounced metachromatic effects than with either more acid or more alkaline buffer solutions.     

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     

Cytochemical Method to Localize Acidic Nuclear Proteins

Fast green FCF was used to localize acidic nuclear proteins in sections of young flower buds of Limnophyton obtusifolium (L.) Miq. After extracting nucleic acids, the slides were stained at hydrogen ion concentrations ranging from pH 2.6 to 9.0. At pH 5.0 and 8.0 staining is confined to the nucleus with no cytoplasmic reaction. Staining intensity is greater at pH 5.0 than at pH 8.0. The proteins responding to fast green at pH 8.0 are basic proteins. The positive reaction at pH 5.0 is attributed to acidic nuclear proteins. These findings are confirmed by control preparations. Acetylated slides and slides treated with 0.25 N HCl were unstained at pH 8.0 but staining at pH 5.0 was undisturbed. Dilute alkali (0.003 N NaOH) reduced staining intensity at pH 5.0 but had no effect at pH 8.0. Methylated slides did not stain at pH 5.0, but at pH 8.0 staining was unaffected. Deamination blocked staining at both pH's. It is concluded that fast green at pH 5.0 specifically binds with acidic nuclear proteins.     

A Nissl Method Using Buffered Solutions of Thionin

Experiments were performed in an attempt to obtain a rapid method for staining the chromophilic substance of formalin-fixed nerve cells differentially without resorting to over-staining and subsequent acid-decolorizing. A satisfactory procedure using thionin in dilute buffered solutions was developed: Prepare a stock solution of the dye using 1 g. in 100 ml. of distilled water. Prepare veronal-acetate buffers (Michaelis, 1931) in the range of pH 5 to pH 3.S. To each 10 ml. of the buffer add 0.5 ml. of the stock dye solution. After rinsing in CO₂-free distilled water place mounted or unmounted sections in this solution. (Freshly fixed material, 10μ to 20μ thick, is completely stained in 10 to 20 minutes but over-staining does not occur when longer times are allowed.) Return sections to distilled water (2 changes) and wash until diffusion of excess dye is no longer visible. Wash in 70% ethyl alcohol (2 changes) until diffusion of excess dye is no longer visible. Dehydrate in 95% ethyl alcohol and normal butyl alcohol, clear and mount.

Optimum staining of chromophilic material occurs at pH 3.65. Glial processes are well stained at pH 4.6. Nissl bodies and glial cytoplasm are purplish blue, nuclear chromatin is blue, background is clear at pH 3.65 but pale blue at pH 4.9.     

Selective staining of nucleic acids by osmium-ammine complex in thin sections from lowicryl-embedded samples

Osmium-ammine (OA)/SO2 selectively contrasted RNA- and DNA-containing structures in thin sections from Lowicryl-embedded samples. No cell structures were stained after Epon embedding. RNAse and DNAse digestion experiments demonstrated that only RNA and DNA were stained in Lowicryl thin sections. Protease digestion did not modify the staining reaction. The very fine end-reaction produced a very high resolution of the stained structures. The staining reaction was not due to the presence of SO2 but to the low pH of the solution (ranging from 1.5-2.2). OA in glycine buffer, pH 1.5, selectively contrasted nucleic acids. Electrostatic bonds between nucleic acids and OA complex were probably involved in the staining reaction. Increasing the pH value of the staining medium resulted in loss of OA specificity for nucleic acids. The high electrolyte concentration of the staining medium hindered the staining reaction.     

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.     

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.     

Myofibrillar ATPase histochemistry of rat skeletal muscles: a "two-dimensional" quantitative approach

In histochemical investigations of skeletal muscle, the fibers are commonly classified into three types according to their staining for myofibrillar ATPase (mATPase). In serial sections of skeletal muscles from normal Wistar rats, we compared two common staining methods for mATPase: (a) an ac-ATPase technique, with pre-incubation at pH 4.7, and (b) a fixed alk-ATPase technique, using treatment with 5% paraformaldehyde followed by pre-incubation at pH 10.4. In addition, the same fibers were stained in subsequent serial sections for succinate dehydrogenase (SDH) activity. Staining intensities were objectively evaluated by microphotometric measurements of optical density. Combining both mATPase methods in consecutive serial sections ("two-dimensional approach") led to the identification of four distinct clusters of fibers: Types I, IIA, and two subgroups of Type IIB, as separated by their staining densities for fixed alk-ATPase (IIBd dark, IIBm moderate). The mean intensity of SDH staining per fiber type, as measured in the central core of the fibers, was ranked such that IIA greater than I greater than IIBd greater than IIBm. The analyzed muscles (tibialis anterior, biceps brachii) were markedly heterogeneous with respect to the topographic distribution of different fiber types. In comparison to other muscle portions, the regions containing Type I fibers ("red" portions) showed a higher IIBd vs IIBm ratio and more intense SDH staining for either subtype of the IIB fibers. The IIBd fibers probably correspond to the Type 2X fibers of Schiaffino et al.     

Methods for the Demonstration of Lipid Applied to Compact Bone

Sections of compact bone were cut from the diaphysis of the femur, tibia, and humerus from dogs and monkeys. These sections were either ground thin and decalcified, or decalcified and subjected to frozen sectioning. Decalcification of the sections was effected by immersion in either Decal, 10% formic acid, 10% formic acid-sodium citrate (pH 4.5) or 20% aqueous EDTA. Sections were routinely stained with oil red O, Sudan black B, or Fettrot 7B. In addition, Nile blue A and phosphine 3R were also employed. Sections stained with phosphine were viewed with a fluorescence microscope. Control sections were extracted with lipid solvents prior to application of the staining procedures. The results indicate that lipid is present in compact bone within the osteocytes, lacunae, canaliculi, and organic matrix. The significance of the lipid in these sites, particularly extracellularly, is unknown.     

An improved method for preparing cryostat sections of undecalcified bone for multiple uses.

We developed a technique that permits the use of serial sections (7-20 microns) from a single fixed piece of bone tissue for immunofluorescence, measurement of fluorescent bone labels, enzyme histochemistry, and general staining. This technique combines modifications of previously established methods with perfusion of the polymer polyvinylpyrrollidone (PVP) to improve sectioning, and produces reliable sections with good preservation of both hard and soft tissues. The combination of techniques from several workers, the use of perfusion with a polymer to increase the sectionability of the bone, and the addition of a gelatin adhesive on top of pressure-sensitive adhesives represent a significant improvement over previously described methods. The sections obtained are usable for immunocytochemistry, general staining, enzyme histochemistry, and visualization of fluorescent bone labels. We have consistently used tissues prepared in this manner for immunohistochemical demonstration of neuropeptides in skeletal tissues and for localizing tartrate-resistant acid phosphatase (TRAP). In addition, other tissues obtained from PVP-perfused rats, such as brain, spinal cord, muscle, gut, and sympathetic ganglia, are also well preserved and demonstrate immunohistochemical staining comparable to and possibly superior to that obtained with normal fixation protocols.     

Effects of orthodontic tooth movement on the Alcian blue staining patterns of rat alveolar bone: an histochemical study

There is little information available concerning the effects of orthodontic forces on glycosaminoglycans (GAG) of alveolar bone. The present study identifies changes in Alcian blue staining intensity in rat alveolar bone undergoing resorption resulting from a heavy (25g) tipping force applied to the adjacent teeth by a separating spring. One day after force application, bone from treated animals (internal control and experimental sides) demonstrated more intense staining with Alcian blue, pH 1.0 (p less than 0.005) and pH 2.5 (p less than 0.05) than external controls (untreated animals). By day 3, the intensity of Alcian blue staining of treated alveolar bone was similar to untreated. Chondroitinase AC, ABC and testicular hyaluronidase predigestion did not completely block the staining reaction, suggesting that both GAG and noncollagenous proteins were demonstrated. Mean cross-sectional areas of the interdental septum of the experimental side were nearly 44% less than that of the internal control side after 3 days and nearly 62% less after 5 days. The study suggested that alterations in bone GAG levels occurred prior to tooth movement as histochemical changes occurred after force application but before initiation of significant septal resorption. A precise appraisal of the types of macromolecules effected awaits future biochemical analysis. The results of the present work strongly suggest the use of an external control group for future studies, as Alcian blue staining reactions of the internal control side of treated animals were not similar to those of external controls.