Automated differential staining for cartilage and bone in whole mount preparations of vertebrates

An automated, rapid procedure for differential staining of cartilage and bone of vertebrates is described. The process involves rapid, complete staining of freshly skinned, eviscerated specimens after 30 sec immersion in a 70 C water bath, fixation in formol acetic alcohol and a rinse in 70% alcohol. Using an automatic tissue processor, the specimen is stained in alcian blue for 24 hr and macerated in 3% potassium hydroxide for 8 hr. Staining in alizarin red with maceration in 3% potassium hydroxide is completed manually. The specimens are cleared and preserved in glycerol. Good quality evenly stained specimens can be examined in less than three days and up to 600 fetuses can be processed in less than five days.     

A modified differential stain for cartilage and bone in whole mount preparations of mammalian fetuses and small vertebrates

Fixation in formol-acetic-alcohol as a prelude to the staining of whole mount vertebrate skeletons with alcian blue and alizarin red S has greatly facilitated the enzyme clearing step of the method outlined by Dingerkus and Uhler. The modified method has been tested on fetal and neonatal mice, and on a variety of vertebrates including bony fish, reptiles, amphibia and birds, and shown to be rapid, reproducible and permanent. The method is not so rapid as that reported by Kimmel and Trammell but is superior at least in certain circumstances. In the present study, optimal results were obtained by fixing in formol-acetic-alcohol for 40 minutes, staining cartilage with alcian blue 8GX, then clearing with trypsin. The time taken to complete the latter step was reduced significantly by incubation at 37 C. The next step was to stain bone using alizarin red S in a weak solution of potassium hydroxide, followed by clearing in a potassium hydroxide-glycerol series.     

A rapid procedure for routine double staining of cartilage and bone in fetal and adult animals

A simple, rapid procedure for dual staining of cartilage and bone in rodents, particularly in late gestation, has been developed for routine use. The procedure involves rapid, complete skinning of fresh eviscerated specimens following a 30 sec immersion in a 70 C water bath. The unfixed specimen is stained in a mixture of 0.14% Alcian blue and 0.12% alizarin red S in ethanol and glacial acetic acid. Specimens are then macerated in 2% KOH, cleared and hardened in 1:1 glycerin and distilled water, and stored in pure glycerin. Rapid staining of cartilage only is done in a mixture of 0.08% Alcian blue, glacial acetic acid, and ethanol, with subsequent maceration, clearing, and hardening as in the double staining procedure. Rapid staining of bone only, concurrent with maceration of soft tissue, can be done by placing fresh, unskinned specimens in a diluted mixture of alizarin red S in 2% KOH, with subsequent clearing and hardening in 1:1 distilled water and glycerin. Good quality fetal specimens can be prepared for examination by any of these procedures in a minimum of 11/2-2 days as compared to a minimum of 4-5 days for other procedures. Double stained specimens can be examined for abnormalities of the cartilage as well as bone.     

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.     

Differential staining of cartilage and bone in whole mouse fetuses by alcian blue and alizarin red S

The procedure described by Inouye ('76) for the staining of full-term mouse fetal skeletons has been adapted for use with mouse embryos and fetuses of days 14-18 of gestation. The main adaptations for younger specimens involve a longer time in acetone, in lieu of skinning, and omission of the aqueous KOH step. These adaptations require more time but result in consistently good staining of intact specimens.     

En bloc staining of articular cartilage and bone

Blocks of canine and porcine articular cartilage were stained en bloc with Weigert's iron hematoxylin or Harris' hematoxylin with or without eosin Y counterstaining and cleared in methyl salicylate. The morphology and three-dimensional relationships of chondrocytes were best demonstrated with Weigert's iron hematoxylin. The morphology of the cartilage and chondrocytes was superior to that in sections of routine hematoxylin and eosin stained, paraffin processed samples. The three-dimensional localization of intracellular lipids in individual and clones of chondrocytes was observed when cartilage samples were stained with oil red O and mounted directly in a water-based medium. Blocks of decalcified bone were stained en bloc with Weigert's iron hematoxylin and cleared with methyl salicylate. The three-dimensional orientation of osteocytes around osteonal canals, in circumferential lamellae, and in interstitial lamellae was demonstrated. The morphology of "cutting cones" in cortical bone also was observed.     

A modified mallory-cason staining procedure for large cryosections.

The classic Mallory-Cason staining procedure has been modified for application to sections "on tape" obtained from large deep frozen tissue specimens. These 20 microns cryosections are collected on tape from a large heavy duty cryomicrotome. The stained sections provide anatomical details that are not revealed by other techniques. The merit of this procedure is found in the support of modern medical modalities, both for research and educational purposes.     

Whole-mount bone and cartilage staining of chick embryos with minimal decalcification

Abstract

Whole-mount staining with Alcian blue for cartilage and alizarin red for bone has been widely used for visualizing the skeletal patterns of embryos and small adult vertebrates. The possibility of decalcification by the acidic Alcian blue solution is known, but standard staining protocols do not always avoid this issue. We investigated the effects of acidity on the stainability of developing bones in stage 36 chick embryos and developed an optimal procedure for obtaining reliable results with minimal decalcification. The diaphyses of long bone rudiments and the maxillofacial membranous bones progressively lost their stainability with alizarin red when the chick embryos were soaked for long periods in the preceding acidic Alcian blue staining solution for cartilage. Unless the acidity was neutralized with an alkaline solution, the remaining acidity in the specimens rendered the pH sufficiently low to prevent the subsequent alizarin red staining of the bones. These findings indicate that the mineralizing bones at the early stages of development are labile to acidity and become decalcified more substantially during the staining process than previously appreciated. The following points are important for visualizing such labile mineralizing bones in chick embryos: 1) fixing with formaldehyde followed by soaking in 70% ethanol, 2) minimizing the time that the specimens are exposed to the acidic Alcian blue solution, and 3) neutralizing and dehydrating the specimens by an alkaline-alcohol solution immediately after the cartilage staining. When the exact onset and/or an early phase of ossification are of interest, the current double-staining procedure should be accompanied by a control single-staining procedure directed only toward bone.     

A rapid and specific fluorescent activity staining procedure for transamidating enzymes.

A rapid, sensitive, and specific procedure has been developed for detection of transsamidatingenzymes (transglutaminase, e.g., factor XIII) after agarose gel electrophoresis. The technique is based on the transamidase-catalyzed incorporation of the fluorescent monodansylthiacadaverine into casein. The high sensitivity enables detection and characterization of transamidases in blood plasma, platelet, and red blood cell lysate and tissue extracts. The technique can also be combined with crossed immunoelectrophoresis.     

Aldehyde fuchsin staining of pancreatic B cells. Reproducible high-contrast staining of formalin-fixed and paraffin-embedded material

Summary The use of Aldehyde Fuchsin for the demonstration of B cell granules in the pancreatic islets on material fixed in formalin and embedded in paraffin has led to variable results.Treatment of such sections for 1 h with Bouin's fluid or 5% glutaraldehyde prior to deparaffinization, however, stabilizes the secretory granules in B cells. In addition, the zymogenic granules of the acinar cells exhibit increased stainability with the permanganate Aldehyde Fuchsin procedure.     

Fast green staining of whole embryos for examination and photography.

Immersion for 15 seconds in a 1:1 solution of 0.25% fast green and 95% ethyl alcohol facilitates the examination of normal and abnormal embryos for gross surface morphology and renders their surface detail highly photogenic. The method does not interfere with subsequent histological staining when the embryos are sectioned.