A Modified One-Step Trichrome Stain for Demonstration of Fine Connective Tissue Fibers

Gomori's one-step trichrome procedure was modified to improve coloration of fine connective tissue fibers. Paraffin sections from tissues fixed in alcohol, acetone, Zenkerformol, 10% formalin, Kaiserling's or Carnoy's fluid were mordanted 1 hr at 56 C in Bouin's solution, stained 1 min in a trichrome solution (chromotrope 2R-phosphomolybdic acidaniline blue WS) adjusted to pH 1.3 with HCl, rinsed in 1% aqueous acetic acid, dehydrated and covered. Collagen, reticulum fibers, basement membranes, ring fibers around splenic sinuses, intercalated discs in cardiac muscle and cartilage were colored blue. Nuclei, cytoplasm, fibrin, muscle fibers and elastic fibers were stained red. Pretreatment of sections with Bouin's solution enhanced the affinity of tissues for chromotrope 2R and was found essential for satisfactory coloration of material fixed in alcohol, acetone, formalin or Carnoy's fluid. Because this method does not require differentiation, it gave uniform results even in the hands of inexperienced laboratory trainees. No fading was observed in sections stored for more than 8 yr.     

Processing techniques for the demonstration of myelin basic protein in paraffin-embedded optic nerve: an immunoperoxidase study of the developing albino rat

A comparison was made of the effects of various fixation and processing conditions upon the antigenicity of myelin basic protein (MBP) in sections of paraffin-embedded optic nerve from the developing albino rat as judged by the unlabeled peroxidase-antiperoxidase technique. The fixatives used were: Perfix, 4% and 2% buffered paraformaldehyde (pH 7.4), 10% buffered formalin (pH 7.4); Bouin's, Clark's, and Carnoy's fixatives, and 20% formalin in a solution of HgCl2 that had been saturated at 1 degrees C. Perfix appeared to be the best fixative for the preservation of morphology and MBP antigenicity during the early stages of myelinogenesis but was not satisfactory during the later stages. The buffered aldehydes were slightly more destructive of MBP antigenicity than was Perfix, but they produced satisfactory results following the first postnatal week. Bouin's fixative was similar in effect to the buffered aldehydes, but nonspecific background staining was higher. HgCl2/formalin, Clark's and Carnoy's fixatives were unsuitable. No differences were noted in staining between material processed for embedding using 5, 30, or 60 min schedules.     

Polyacrylamide gel electrophoresis and immunoblotting of proteins extracted from paraffin-embedded tissue sections

We show in this communication that polyacrylamide gel electrophoresis (PAGE) and immunoblotting of proteins can be performed using one to two 5-7 micron paraffin sections of tissues fixed in non-cross-linking fixatives (acetone, alcohol, or modified Carnoy's solution). Proteins for study were extracted from paraffin sections of mouse foot pad and liver. The presence of unaltered keratin polypeptides in tissues fixed with either acetone or alcohol was demonstrated in gels stained with Coomassie brilliant blue. The preservation of their antigenic determinants was demonstrated with immunoblotting. Furthermore, the immunoreactivity of soluble proteins, such as albumin, remained unaltered in immunoblots obtained from paraffin-embedded mouse liver sections. These data indicate that tissues embedded and stored in paraffin are useful for the above-mentioned biochemical and immunological studies and may therefore be an important technique for diagnostic pathology or retrospective studies.     

Application of the Papanicolaou Stain to Paraffin Sections

Routine paraffin sections from tissues fixed either in aqueous formalin, 80% alcohol (with or without 1% trichloracetic acid added), Carnoy's alcohol-chloroform-acetic (6:3:1) and Bouin's fixative were stained as follows: Harris' hematoxylin, 6 min; running water, 2-3 min; ascending grades of alcohol to 95%; orange G, 0.5% and phosphotungstic acid, 0.015% in 95% alcohol, 5 min; 95% alcohol, 2 changes; Papanicolaou's EA36, 2.5 min; dehydration, clearing, and covering in Permount. The results show morphology better than hematoxylin and eosin and the technic is recommended particularly for keratin, which always stains bright orange.     

Optimal immunoreactivity of keratin proteins in formalin-fixed, paraffin-embedded tissue requires preliminary trypsinization. An immunoperoxidase study of various tumours using polyclonal and monoclonal antibodies

The effect of preliminary trypsinization on the immunoreactivity of keratin proteins in formalin-fixed, paraffin-embedded tissues of a variety of tumors (squamous cell carcinomas, adenocarcinomas, mesotheliomas, and transitional cell carcinomas) was evaluated. Three types of trypsin (Type II and Type IX porcine trypsin and Type III bovine trypsin) and varying concentrations of trypsin were assessed. Immunoreactivity of keratin proteins was determined using rabbit anti-keratin antibodies and monoclonal antibodies (combination of AE1 and AE3) and immunoperoxidase techniques. Preliminary trypsinization was mandatory for optimal immunoreactivity of keratin proteins using either polyclonal or monoclonal antibodies. Excellent results were obtained using Type II porcine trypsin at concentrations of 25 mg/dl for 30-45 min or 50 mg/dl for 20 min, at 37 degrees C. Trypsin treatment with excessive concentrations of enzyme and/or extended incubation times promoted tissue digestion and in some cases, yielded decreased immunoreactivity and altered staining patterns.     

A Method for the Selective Removal of Deoxyribonucleic Acid from Tissue Sections

The deoxyrihonucleic acid (DNA) of chromatin undergoar depurinization on mild acid hydrolysis with a picric acid-formaldehyde mixture (Bouin's fluid). The apurinic acid thus formed is degraded by condensation with aniline and is lost from tissue sections, but ribonucleic acid (RNA) in nucleoli and cytoplasm is well preserved. Technique: Fi in Carnoy's fluid (ethanol:acetic acid 3:1 or ethanol:chloroform:acetic acid 6:3:1) or in aldehydes (10% formalin or 2.5% glutaraldehyde bsered to pH 7.0). Hydrolyse deparaEnii sections 12-24 hr at 27-50 C in Bouin's fluid, wash in distilled water, immerse in 25% (v/v) acetic acid, treat 1 hr at 27-30 C with 10% (v/v) dine in 25% acetic acid, wash in 25% acetic acid and then in water. Stain 10-40 min with 0₃% toluidine blue in 0.05 M potassium biphthalate bder (pH 4.0); rinse in distilled water, pass to 10% (w/v) ammonium molybdate for 1 min, rinse again in water and pass through tert-butanol and xylene to a synthetic resin. Chromatin and chromosomes are pale green; RNA in nucleoli and cytoplasm deep purple.     

Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion.

A new procedure is described to generate single-stranded DNA by exonuclease III (Exo III) digestion for bromodeoxyuridine (BrdU) immunocytochemistry on tissue sections. We compared this procedure with the most widely used procedure of DNA denaturation with 2 N HCl. In vivo and in vitro pulse and continuous labelling of tissues and cells were used. The specimens were fixed in formalin, ethanol, glutaraldehyde, Carnoy's, Bouin's or Zamboni's fixative and embedded in paraffin or used unfixed as cryostat sections or cytospin preparations. After Exo III digestion, BrdU substituted DNA was detected irrespective of the fixation procedure applied. The optimal protocol for nuclease digestion appeared to be simultaneous incubation, of 10 Units Exo III per ml EcoRI buffer and anti-BrdU monoclonal antibody at 37 degrees C. The advantages of Exo III digestion for BrdU immunocytochemistry compared to acid denaturation were: less non-specific nuclear background reactivity, no DNA renaturation, less DNA loss, optimal nuclear morphology, increase in antibody efficiency and the possibility for simultaneous detection of acid-sensitive tissue constituents. Disadvantages of the Exo III digestion are decreased sensitivity and the need for more rigorous pepsin pretreatment. We conclude that Exo III digestion of DNA is an appropriate alternative for acid denaturation for BrdU immunocytochemistry on sections of pulse-labelled specimens.     

The importance of tissue fixation for light microscopic immunohistochemical localization of peroxisomal proteins: the superiority of Carnoy's fixative over Baker's formalin and Bouin's solution

We have compared the effects of fixation with three commonly used fixatives upon preservation of the antigenicity of six peroxisomal proteins in rat liver using both immunohistochemical staining and Western blotting of fixed tissue extracts. The immunoreactivity of all six peroxisomal proteins was well preserved and peroxisomes were clearly identified in material fixed in Carnoy's fixative. Moreover, the corresponding proteins stained well in Western blots prepared from extracts of Carnoyfixed material. The intensity of the immunohistochemical staining was reduced at different rates for individual peroxisomal proteins after fixation in Baker's formalin, but peroxisomes were still well visualized with antibodies to catalase and some -oxidation enzymes. No evidence of immunohistochemical staining for any peroxisomal antigens was obtained after fixation in Bouin's fluid. For detection of the antibody binding sites in Carnoy's fixed material, the avidin-biotin-peroxidase complex (ABC) with aminoethyl carbazole as chromogen was found to be superior to the methods of peroxidase-antiperoxidase/diaminobenzidine and protein A-gold with silver intensification. Using Carnoy-fixative and the ABC-method, we demonstrate light microscopic immunohistochemical localization of peroxisomal antigens in several rat tissues as well as in human post-mortem liver.     

Use of immunocytochemical techniques for the localization of human placental lactogen.

The recent claim by Gau and Chard (Br J Obstet Gynaecol 83:876, 1976) that, on theoretical grounds, it may be impossible to demonstrate the presence of human placental lactogen in placental tissue using the immunoperoxidase technique, has been reinvestigated. Placental tissue fragments fixed in Carnoy's fluid retained their morphologic identity compared with tissue fixed in formalin. Using these nonformalin fixed tissues, human placental lactogen was successfully localized within the cytoplasm of the syncytial layer of the placental villus. It is concluded that placental villi at term do in fact contain sufficient human placental lactogen to be demonstrated using the immunoperoxidase technique in contrast to the observation of Gau and Chard.     

Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.

We describe a new approach for retrieval of antigens from formalin-fixed, paraffin-embedded tissues and their subsequent staining by immunohistochemical techniques. This method of antigen retrieval is based on microwave heating of tissue sections attached to microscope slides to temperatures up to 100 degrees C in the presence of metal solutions. Among 52 monoclonal and polyclonal antibodies tested by this method, 39 antibodies demonstrated a significant increase in immunostaining, nine antibodies showed no change, and four antibodies showed reduced immunostaining. In particular, excellent immunostaining results were obtained with a monoclonal antibody to vimentin as well as several different keratin antibodies on routine formalin-fixed tissue sections after pre-treatment of the slides with this method. These results showed that after antigen retrieval: (a) enzyme predigestion of tissues could be omitted; (b) incubation times of primary antibodies could be significantly reduced, or dilutions of primary antibodies could be increased; (c) adequate staining could be achieved in long-term formalin-fixed tissues that failed to stain by conventional methods; and (d) certain antibodies which were typically unreactive with formalin-fixed tissues gave excellent staining.     

In vitro biosynthesis of rat sperm outer dense fiber components

Conditions were established for in vitro culture of seminiferous tubules of adult rat testis. Tubules fragments were able to incorporate radioactive amino acids for up to 6 hours of incubation at 32 degrees C in a modified Eagle's minimum media, indicating biosynthetic activity. Addition of D-glucose (11 Mm) increased the incorporation of either [3H] Leucine or [35S] Methionine four-fold in the protein components of seminiferous tubules. Polyclonal antibodies against outer dense fibers (ODF) polypeptides, which represent approximately 30% of the total sperm proteins, immunoprecipitated 5% of the total radioactivity from cultures carried out either in the presence or absence of D-glucose. Moreover, antibodies specific for the 27-30 kilodalton polypeptides of ODF immunoprecipitated 2% of the total radioactivity, showing no differences in the presence and absence of D-glucose. This study indicates that ODF polypeptides can be synthesized in vitro at 32 degrees C with and without D-glucose.     

Effect of fixation in Carnoy's solution on nucleic acid and protein concentration in the rabbit superior cervical sympathetic ganglion]

By means of biochemical techniques, the stability of RNA, DNA and protein contents has been demonstrated in the rabbit superior cervical sympathetic node fixed in Carnoy's solution for 2 hours at 4 degrees C. A 2 hour fixation in Carnoy's fluid at room temperature, results, however, in a considerable loss of RNA (21%). The loss of DNA and protein did not exceed 2% of the total amount of the fresh ganglion tissue. Fixation at a higher temperature (37%) increased the loss percentage protein and of nucleic acids: 3.1, 5.5 and 42%, for protein, DNA and RNA, resp.     

Lime treatment of keratinous materials for the generation of highly digestible animal feed: 1. Chicken feathers

Chicken feather keratin was treated with lime (calcium hydroxide) to obtain a liquid product rich in amino acids and polypeptides that can be used as an animal feed supplement. The effect of treatment conditions and the properties of the soluble keratin were studied. At high temperatures (150 degrees C), 80% of feather keratin was solubilized within 25 min, whereas a relatively longer reaction time (300 min) is needed at moderate temperatures (100 degrees C). After 3h of hydrolysis at 150 degrees C, 95% of feather keratin was digested. For the recommended conditions (100 degrees C, 300 min, and 0.1g Ca(OH)(2)/g dry feather), after lime treatment, about 54% of calcium can be recovered by carbonating. In rumen fluid, ammonia production from soluble keratin was similar to that of soybean and cottonseed meals and was greatly less than that of urea, indicating that no ammonia toxicity will result from cattle being fed soluble keratin.     

Change of hepatitis B virions (Dane particles) phosphorylation pattern by human hepatoma cell particulate fraction

Protein kinase activity has been found in hepatitis B virions (Dane particles) purified from the plasma of hepatitis B surface antigen carriers [Albin, C., and Robinson, W.S. (1980) J. Virol. 34, 297-302]. Dane particles were purified from the pooled, HBeAg-positive plasma. When this preparation was incubated with [gamma 32P]ATP in the presence of 10mM MnCl2 and 0.5% NP-40 for 15 seconds at 30 degrees C, several phosphorylated polypeptides of 20,000, 42,000, 48,000, 50,000 and 56,000 daltons were detected in sodium dodecyl sulfate-polyacrylamide gels. When the Dane particles were incubated with [gamma 32P]ATP, 10 mM MnCl2, and 0.5% NP-40 in the presence of human hepatoma cell (J-5) particulate fraction at 30 degrees C, 15 seconds, the 42,000, 48,000 and 50,000 daltons phosphorylated polypeptides were not found. When human peripheral blood lymphocytes particulate fraction was incubated with Dane particles under the same conditions, no change of Dane particle phosphorylated polypeptides was detected. Previous publications [Albin, C., and Robinson, W.S. (1980) J. Virol. 34, 297-302; Gerlich, W.H. et al. (1982) J. Virol. 42, 761-766] showed that when hepatitis B core particles purified from hepatoma tissues contained protein kinase activity, only phosphorylated polypeptide was 20,000 daltons. Our data suggested that when Dane particles were put in an environment of hepatoma cells (or tissues), the protein kinase could only phosphorylate selected polypeptides in these particles.     

Glycol Methacrylate in Light Microscopy a Routine Method for Embedding and Sectioning Animal Tissues

Glycol methacrylate (GMA), a water and ethanol miscible plastic, was introduced to histology as an embedding medium for electron microscopy. This medium may be made soft enough for cutting thick sections for routine light microscopy by altering its composition. A procedure for the infiltration, polymerization, and sectioning of animal tissues in GMA for light microscopy is presented which is no more complex than paraffin techniques and which has a number of advantages: (I) The GMA medium is compatible with both aqueous fixatives (formaldehyde, glutaraldehyde, Bouin's, and Zenker's) and non-aqueous fixatixes (Carnoy's, Newcomer's, ethanol, and acetone). (2) Undue solvent extraction of the tissue is avoided because adequate dehydration occurs during infiltration of the embedding medium. Separate dehydration and clearing of the tissue prior to embedding is eliminated. (3) When polymerized, the supporting matrix is firm enough that hard and soft tissues adjacent to one another may be sectioned without distortion. (4) Thermal artifact is reduced to a minimum during polymerization because the temperature of the tissue may be maintained at 0-4 C from fixation through ultraviolet light polymerization of the embedding medium. (5) Shrinkage during polymerization of the embedding medium is minimized by prepolymerization of the medium before use. (6) Sections may be easily cut using conventional steel knives and rotary microtomes at a thickness of 0.5 to 3.0 microns, thus improving resolution compared with routinely thicker paraffin sections. (7) The polymerized GMA medium is porous enough so that staining, auto radiography, and other histological procedure are done without removal of the embedding medium from the sections. A list of these stains and related procedures are included. (8) Enzyme digestion of ultra thin sections of tissue embedded in GMA is common in electron microscopic cyto chemistry. me same digestion techniques appear compatible with the thicker seaions used in light microscopy.     

Methacarn fixation--effects of tissue processing and storage conditions on detection of mRNAs and proteins in paraffin-embedded tissues

In this study, we examined suitable conditions for tissue fixation with methacarn and ethanol dehydration and storage of paraffin-embedded tissues (PETs) on gene expression analysis. With fixation and dehydration of rat liver tissues for up to 16 h (overnight) and 1 week, respectively, at 4 degrees C, integrity of extracted total RNAs and polypeptides did not vary, the former integrity being constantly lower than that with unfixed frozen tissue, while protein yield was slightly reduced with increasing dehydration. Retained expression levels of mRNAs and proteins were mostly unaffected by the period of fixation but slightly fluctuated with the length of dehydration. When PETs were stored for up to 12 months, integrity of both total RNAs and polypeptides was retained at 4 degrees C but reduced at room temperature. Reduced expression levels of mRNAs and proteins were also noted by storage at room temperature after 12 and 3 months, respectively. However, neither tissue processing nor storage affected variability in either mRNA or protein levels among samples. Thus, the results suggest that, for gene expression analysis, tissues can be fixed with methacarn and dehydrated for at least 1 day and 1 week, respectively, and PETs can be stored for at least 12 months, but a temperature of 4 degrees C is preferable.     

Preliminary evaluation of mast cells and angiogenesis processes in experimental fibrosarcoma

The aim of the study was the evaluation of angiogenesis processes in fibrosarcoma induced by 3-methylcholanthrene (3-Mc) in reference to the number of mast cells (MCs). 76 male Wistar rats were divided into 4 groups: two experimental (E) groups--after injection of 0.2 mg 3-Mc dissolved in olive oil (0.25ml), and 2 control (C) groups. In E1 group, 52 rats were killed after development of the fibrosarcoma; E(2)--10 rats were killed before development of the tumor; C(1)-8 rats received 0.25ml olive oil; C(2)--8 rats received no treatment. Tissue material was fixed in buffered formalin or Carnoy's and Bouin's fluid. Paraffin sections were stained with H+E and Azan methods, and with alcian blue-saphranine and toluidine blue. Immunohistochemical reactions detecting tryptase in MCs were also performed. Angiogenic objects (microvessels and single endothelial cells) were recognized using antibodies against factor VIII (vWF), P selectin (CD-62P), and CD-90. We found a distinct relationship between intensification of neoangiogenesis at the tumor periphery and increased number of MCs.     

In vivo thermal conductivity of the human forearm tissues

The effective thermal conductivities of the skin + subcutaneous (keff skin + fat) and muscle (keff muscle) tissues of the human forearm at thermal steady state during immersion in water at temperatures (Tw) ranging from 15 to 36 degrees C were determined. Tissue temperature (Tt) was continuously monitored by a calibrated multicouple probe during a 3-h immersion of the resting forearm. Tt was measured every 5 mm from the longitudinal axis of the forearm (determined from computed-tomography scanning) to the skin surface. Skin temperature (Tsk), heat loss (Hsk), and blood flow (Q) of the forearm, as well as rectal temperature (Tre) and arterial blood temperature at the brachial artery (Tbla), were measured during the experiments. When the keff values were calculated from the finite-element (FE) solution of the bioheat equation, keff skin + fat ranged from 0.28 +/- 0.03 to 0.73 +/- 0.14 W.degrees C-1.m-1 and keff muscle varied between 0.56 +/- 0.05 and 1.91 +/- 0.19 W.degrees C-1.m-1 from 15 to 36 degrees C. The values of keff skin + fat and keff muscle, calculated from the FE solution for Tw less than or equal to 30 degrees C, were not different from the average in vitro values obtained from the literature. The keff values of the forearm tissues were linearly related (r = 0.80, P less than 0.001) to Q for Tw greater than or equal to 30 degrees C. It was found that the muscle tissue could account for 92 +/- 1% of the total forearm insulation during immersion in water between 15 and 36 degrees C.     

Schizogony in Haemoproteus columbae Kruse.

To fill in some of the gaps in our knowledge of Schizogony of Haemoproteus columbae Kruse, transmission experiments involving inoculation into pigeons (Columba livia Gmelin) of sporozonites from salivary glands of the hippoboscid fly Pseudolynchia canariensis (Macquart) were carried out. We were unable to detect prepatent schizonts or to observe schizogonic development when infection became chronic. Schizonts were mainly confined to lung tissue. Observations of parapatent schizonts were made in smears and tissue sections. A variety of forms was found. Cytomeres were rarely encountered. Two types of morphologically distinct merozoites were seen. One type was twice as large as large as the other and was thought to repeat the process of schizogony several times before invading erythrocytes. Schizonts with cytoplasmic clefts were not common in our material due to the fixatives used (Bouin's and Carnoy's). Merozoites were occasionally observed inside monocytes, probably being phagocytosed.     

The identification and partial characterization of merosin--a new specific protein of the basement membranes of certain tissues

A tissue-specific basement membrane-associated protein has been identified by the use of monoclonal antibodies prepared against a protein fraction of human placenta. In frozen sections of human tissues the monoclonal antibodies decorated basement membranes of Schwann cells, striated muscle, and trophoblast. In antibody-affinity chromatography of limited pepsin digests of human placenta, a 65-kDa polypeptide was bound by the monoclonal antibodies. Polyclonal antisera and new monoclonal antibodies were raised against the isolated 65-kDa polypeptide, and they stained human tissues identically to the original monoclonal antibodies. An 80-kDa polypeptide was detected by these antibodies in placental extracts prepared without proteolysis. The 65-kDa and 80-kDa polypeptides were immunologically distinct from laminin, type IV collagen, fibronectin and major serum proteins. These polypeptides are presumably derived from a novel basement membrane-associated protein which we named merosin. Several cDNA clones were isolated which code for a protein specifically recognized by polyclonal antibodies to the 65-kDa fragment. In developing mouse tissues, merosin was first detected at the newborn stage. The restricted tissue distribution and the late development appearance of merosin suggest that the protein has a tissue-specific function in highly differentiated cells.