A Method of Affixing Separate Thick Sections from Materials Embedded in Paraffin

Botanical studies often require thick histological sections (for embryology, pollen and spore arrangement in tetrads, etc.). Study of the original position of the generative cell in Angiosperms, for example (Huynh 1972), requires paraffin sections bearing entire pollen grains with a diameter of up to 80 μm. However, it is impossible to obtain ribbons with sections of such thickness. If the sections are affixed separately, they do not hold so strongly to slides as do those mounted as ribbons; this difficulty increases with thickness of section. in addition, affixing sections separately with the required order and spacing is tedious and difficult, demands a great deal of time, and even so, is not always successful. the simple method described here can remedy such inconveniences.     

Paraffin Section Thickness—A Direct Method of Measurement

Paraffin section thickness may be directly measured by re-embedding the sections wider consideration, cutting them again at right angles to the original plane of sectioning, and taking direct measurements with a filar micrometer after staining and mounting. Conditions and materials with which relatively un-distorted 3 and 5 μ sections were secured include (a) a hand-honed knife with a 23° facet bevel, set at a clearance angle of 7°, and (b) a hard paraffin (56-58°) embedding medium, preferably with 5% beeswax and 5% bayberry wax added. By taking direct measurements, it was found that bull testis tissue cut at a microtome setting of 10μ averaged 10.82 μ in thickness. Settings of 5 μ and 3 m resulted in sections averaging 5.25 and 3.31 μ in thickness respectively. Stages in sporogenesis of Onoclea sensibilis, Lewitsky fixed, were examined after sectioning at settings of 10, 5, and 3 μ to determine necessity for thin sections. For this material, it was indicated that mitochondrial preparations as thick as 10 μ were worthless, regardless of good fixation and proper staining. Three-micron sections give the best results.     

Circadian Characteristics of Urinary Epinephrine and Norepinephrine from Healthy Young Women in Japan and USA

Clinically healthy diurnally active young adult women were studied during the same season (March) at the Universities of Kyushu (Fukuoka City, Japan) and of Minnesota (Minneapolis, USA), under comparable conditions, except that the habitual diets were not changed. The subjects (20 Japanese and 16 Americans of mixed Caucasian background) were studied over a single 24-hr span. Urine was collected at 4-hr intervals. A circadian rhythm in total urinary norepinephrine excretion showed similar characteristics in Japanese and Americans. In epinephrine excretion, the Japanese women showed a statistically significantly higher amplitude with higher peak values, but no statistically significant difference in the rhythm-adjusted mean. This intergroup difference is strictly time dependent; it does not come to the fore in urine samples covering the nocturnal rest span of the subjects.     

Circadian Characteristics of Urinary Epinephrine and Norepinephrine from Healthy Young Women in Japan and USA

Clinically healthy diurnally active young adult women were studied during the same season (March) at the Universities of Kyushu (Fukuoka City, Japan) and of Minnesota (Minneapolis, USA), under comparable conditions, except that the habitual diets were not changed. The subjects (20 Japanese and 16 Americans of mixed Caucasian background) were studied over a single 24-hr span. Urine was collected at 4-hr intervals. A circadian rhythm in total urinary norepinephrine excretion showed similar characteristics in Japanese and Americans. In epinephrine excretion, the Japanese women showed a statistically significantly higher amplitude with higher peak values, but no statistically significant difference in the rhythm-adjusted mean. This intergroup difference is strictly time dependent; it does not come to the fore in urine samples covering the nocturnal rest span of the subjects.     

Stain Technology: A Simplified Aluminum Stain in Paraffin Sections of Bone from Hemodialysis Patients

A new simplified method has been devised for staining aluminum and has been tested in paraffin sections of bone from 60 patients who have undergone hemodialysis. Iliac crest bone fragments were fixed in 20% phosphate-buffered formalin for less than a day and demineralized at room temperature in 10% phosphate-buffered formalin containing 5% formic acid for only 2 to 3 hr. Four-micron paraffin sections, accompanied by positive controls, were stained with Maloney's aluminum stain, the Berlin blue reaction for iron, dylon or Congo red for amyloid and von Kossa's reaction for calcium. Aluminum and iron were demonstrated particularly at the mineralizing front of bony tissues; aluminum in 52 cases, iron in 45. Dylon staining also gave positive results in 52 cases. It is important in determining whether aluminum deposition is present that the von Kossa reaction remains positive even after demineralization. This method may be more useful for demonstrating aluminum in bony tissues than the complicated and time-consuming resin-embedding method currently used.     

Phosphotungstic Acid-Eosin Combined with Hematoxylin as A Stain for Negri Bodies in Paraffin Sections

Experimentation with the Papanicolaou stain in this laboratory led to the discovery that the eosin, combined with phosphotungstic acid, was responsible for differential staining of Negri bodies. Eosin prepared as in EA 36 was used, but without the light green and Bismarck brown. Paraffin sections of hippocampus from brains of animal affected with rabies were fixed in 10% formol or in a mixture of 2 volumes of saturated aqueous HgCl₂ and 1 volume of absolute alcohol. They were stained first with hematoxylin and then with eosin. This procedure gave better results than staining with other types of eosin or by the original EA 36 mixture. The Negri bodies were well stained and their structure easily visible. The best results were obtained from material fixed with the HgCl₂ solution.     

A Buffer Modification of the Warthin-Starry Silver Method for Spirochaetes in Single Paraffin Sections

The Warthin-Starry¹ procedure, according to Kerr², appears to be -one of the more reliable methods for demonstration of spirochaetes in single paraffin sections. However, the adjustment of the pH of the distilled water used to pH 4.4 with citric acid necessitates the use of indicator each time, and, further, the citric acid stock solution has to be made fresh at frequent intervals because of its susceptibility to mold growth.     

Modified Tetrachrome Method for Osteoid and Defectively Mineralized Bone in Paraffin Sections

A new modification of the tetrachrome method for bone osteoid in paraffin sections has been designed. The modified tetrachrome method suitable for routine use in any histology laboratory retains the simplicity of the original method and gives good results on the freshly fixed, decalcified, paraffin embedded material. Osteoid tissue is stained deep blue and normally mineralized bone is stained red. Defectively mineralized bone stains pale blue or pink and the cellular population is clearly identifiable. The ability to distinguish the osteoid tissue from mineralized bone and connective tissue and cartilage makes diagnosis of osteomalacia or osteoid producing tumors or assessment of ossification process straightforward, without the need for un-decalcified sections. By displaying simultaneously irregularities in the mineralized matrix and morphology of bone cells, the method also permits the diagnosis of conditions recently described in patients with osteoporotic fractures, such as osteocytic degeneration and bone tissue defects.     

Suitability of Different Protein A-Gold Markers for Immunogold-Silver Staining in Paraffin Sections

An incubation protocol to immunolabel Lowicryl semithin sections was applied to paraffin probes. To improve the labeling density, colloidal gold complexes of different preparations and sizes were compared. The type of colloidal gold preparation used was found to affect the specificity of the immunostaining. Gold colloid of 5 nm diameter particle size prepared with white phosphorus minimized nonspecific background labeling of β-casein in paraffin embedded sections of the mammary epithelium of pregnant mice. Gold colloids of 5 nm and 9 nm diameter particle size prepared in varying concentrations of tannic acid generated significant nonspecific staining in similar tissue preparations.     

A Rapid Method for Preparing Paraffin Serial Sections of Teeth and Their Supporting Structures in the Dog

A method of preparing dogs' teeth for serial sectioning involving the following steps is described. The tissues are fixed in 10% formalin followed by partial dehydration in 80% iso-propyl alcohol; decalcified in 5% nitric acid in 10% formalin; washed for 5-8 hours in running water and neutralized in 5% aqueous sodium sulfate for 24 hours, followed by further washing in running water for another 24 hours. They are then dehydrated over a period of 4 days in increasing grades of iso-propyl alcohol and embedded in tissue-mat in sub-atmospheric pressure of 10 lb. Sections so obtained were better suited for cytological study than celloidin preparations, and serial sections could be obtained in a much shorter time.     

Pontine and Cerebellar Norepinephrine Content in Adult Rats Recovering from Focal Cortical Injury

Norepinephrine (NE) plays an important role in motor recovery after brain damage. Most studies concerning NE activity have been performed in the cerebellum, while the role of the pons, the site where the norepinephrinergic locus coeruleus is located, has not yet been elucidated. For this work, we studied the changes in cerebellar and pontine NE content in sham-operated (n = 17), motor cortex injured (n = 6) and recovered rats (n = 12). Motor effects were assessed by means of footprint analysis and sensorimotor evaluation. It was found that after cortical brain damage, the stride length decreases while the stride angle increases after 6 h post-surgery, while the sensorimotor evaluation showed an increase in the motor deficit. Recovery was observed after 24 h. NE content increased in the pons after 6 h and returned to normal levels in recovered rats, with no significant changes observed in the cerebellum. Based on the functional remote inhibition, it is possible that NE exerts an autoinhibitory effect in the pons after motor cortical ablation. On the other hand, the absence of an effect in the cerebellum suggests that cerebellar NE activity related to damage and/or recovery is limited to discrete areas of the structure.     

Glutamatergic Neurotransmission from Melanopsin Retinal Ganglion Cells Is Required for Neonatal Photoaversion but Not Adult Pupillary Light Reflex

Melanopsin-expressing retinal ganglion cells (mRGCs) in the eye play an important role in many light-activated non-image-forming functions including neonatal photoaversion and the adult pupillary light reflex (PLR). MRGCs rely on glutamate and possibly PACAP (pituitary adenylate cyclase-activating polypeptide) to relay visual signals to the brain. However, the role of these neurotransmitters for individual non-image-forming responses remains poorly understood. To clarify the role of glutamatergic signaling from mRGCs in neonatal aversion to light and in adult PLR, we conditionally deleted vesicular glutamate transporter (VGLUT2) selectively from mRGCs in mice. We found that deletion of VGLUT2 in mRGCs abolished negative phototaxis and light-induced distress vocalizations in neonatal mice, underscoring a necessary role for glutamatergic signaling. In adult mice, loss of VGLUT2 in mRGCs resulted in a slow and an incomplete PLR. We conclude that glutamatergic neurotransmission from mRGCs is required for neonatal photoaversion but is complemented by another non-glutamatergic signaling mechanism for the pupillary light reflex in adult mice. We speculate that this complementary signaling might be due to PACAP neurotransmission from mRGCs.     

Circadian Rhythms of Dopamine and Cholecystokinin in Nucleus Accumbens and Striatum of Rats—Influence on Dopaminergic Stimulation

The concentrations of cholecystokinin (CCK) and dopamine (DA) were determined in the nucleus accumbens (anterior, posterior) and striatum of rats every 2 h during a period of 24 h. For both substances, a circadian rhythm was found which was best fitted by a dominant 24-h period superimposed by the second (12 h) and fourth (6 h) harmonics. The rhythms in CCK and DA were negatively correlated because of a difference in phase position by ∼3 h. A dominant DA peak was found in the light phase coinciding with a trough in CCK and vice versa in the dark phase. Based on these data, CCK and DA were determined in rats treated with γ-butyrolactone (GBL; inhibitor of DA release) or thyrotropin-releasing hormone (TRH; stimulator of DA release) at 0900 h or 1300 h to study a putative time-dependency in drug effects. After GBL treatment, CCK as well as DA increased by up to 200%, whereas TRH administration led to a rather complex alteration, inasmuch as CCK was increased or decreased, depending on circadian time, whereas the rhythmic pattern in DA remained relatively unaffected. Comparing the drug effects obtained at 0900 h with the response seen at 1300 h revealed significant quantitative as well as qualitative differences. The results demonstrate that the neurotransmission system investigated changed its level of activity depending on time of day. No changes were obtained that convincingly may be ascribed to colocalization of DA and CCK. It is concluded that the chronobiological data indicate a close interaction of CCK and DA in various areas of rat brain, independent of colocalization.     

Combined Bright Field and Fluorescence Staining of Paraffin Sections for Studying the Fertilization Process in Plants

PAS-toluidine blue O—aniline blue staining of paraffin sections allows study of histological and cytological detail while retaining aniline blue induced fluorescence in all “callose sites”. Because most autofluorescence is eliminated by the PAS-toluidine blue prestaining, the detail and contrast of the fluorescence image is superior to slides stained in aniline blue alone. Slides are stained by the PAS reaction, 0.03% toluidine blue O, alkaline 0.005% aniline blue, and mounted directly in aqueous mounting medium.     

MicroRNAs and Their Targets Are Differentially Regulated in Adult and Neonatal Mouse CD8+ T Cells

Immunological memory, which protects organisms from re-infection, is a hallmark of the mammalian adaptive immune system and the underlying principle of vaccination. In early life, however, mice and other mammals are deficient at generating memory CD8+ T cells, which protect organisms from intracellular pathogens. The molecular basis that differentiates adult and neonatal CD8+ T cells is unknown. MicroRNAs (miRNAs) are both developmentally regulated and required for normal adult CD8+ T cell functions. We used next-generation sequencing to identify mouse miRNAs that are differentially regulated in adult and neonatal CD8+ T cells, which may contribute to the impaired development of neonatal memory cells. The miRNA profiles of adult and neonatal cells were surprisingly similar during infection; however, we observed large differences prior to infection. In particular, miR-29 and miR-130 have significant differential expression between adult and neonatal cells before infection. Importantly, using RNA-Seq, we detected reciprocal changes in expression of messenger RNA targets for both miR-29 and miR-130. Moreover, targets that we validated include Eomes and Tbx21, key genes that regulate the formation of memory CD8+ T cells. Notably, age-dependent changes in miR-29 and miR-130 are conserved in human CD8+ T cells, further suggesting that these developmental differences are biologically relevant. Together, these results demonstrate that miR-29 and miR-130 are likely important regulators of memory CD8+ T cell formation and suggest that neonatal cells are committed to a short-lived effector cell fate prior to infection.     

Staining of the Nucleolus with Protein and RNA Stains for Automatic Measurement of Nucleolar Size in Paraffin Sections

In this paper two staining methods for automatic assessment of nucleolar profile area in paraffin sections of breast carcinomas using the IBAS 2000 image analyzer are reported. In the first method, the nucleolar proteins are stained with phloxine B, in the second the nucleolar RNA is stained with methyl green-pyronin. Both methods are fully reproducible. Statistically, the profile areas observed for the same patient by the two methods were found not to differ significantly. Because of their rigidity and their size in relation to section thickness, nucleoli are particularly attractive cell structures for quantitation, for instance in studies of variables that might be useful in establishing the prognosis for carcinoma patients.     

A Phloxine-Azure-Hematoxylin Sequence for Differential Staining of Cells in Pancreatic Islets

Sections of 6 μ from tissues fixed in Susa or in Bouin's fluid (without acetic acid) and embedded in paraffin were attached to slides with Mayer's albumen, dried at 37 C for 12 hr, deparaffinized and hydrated. The sections fixed in Susa were transferred to a I₂-K₁ solution (1:2:300 ml of water); rinsed in water, decolorized in 5% Na₂S₂O₃; washed in running water, and rinsed in distilled water. Those fixed in Bouin's were transferred to 80% alcohol until decolorized, then rinsed in distilled water. All sections were stained in 1% aqueous phloxine, 10 min; rinsed in distilled water and transferred to 3% aqueous phosphotungstic acid, 1 min; rinsed in distilled water; stained 0.5 min in 0.05 azure II (Merck), washed in water; and finally, nuclear staining in Weigert's hematoxylin for 1 min was followed by a rinse in distilled water, rapid dehydration through alcohols, clearing in xylene and covering in balsam or a synthetic resin. In the completed stain, islet cells appear as follows: A cells, purple; B cells, weakly violet-blue; D cells, light blue with evident granules; exocrine cells, grayish blue with red granules.     

A Staining Sequence for A, B and D Cells of Pancreatic Islets

Recent investigations strongly suggest the elaboration of a third pancreatic hormone by the D cell and the existence of cells which show the staining properties of both B and D cells. Demonstration of these and all other islet cells in a single section is possible by the following staining sequence: (1) of D cells by silver or toluidine blue, (2) of B cells by pseudoisocyanin, and (3) empirical staining of all islet cells together by aldehyde fuchsin, ponceau de xylidine, acid fuchsin and light green. Difficulties in embedding compact pancreatic tissue can be overcome by dehydrating to 80% ethanol, followed by tetrahydrofurane as the intermediate fluid to paraffin infiltration.