Detection of albumin mRNAs in rat liver by in situ hybridization: usefulness of paraffin embedding and comparison of various fixation procedures

Our aim was to define optimal conditions for efficient and reproducible albumin mRNA detection in rat liver by in situ hybridization. We used an albumin-specific [3H]-labeled cDNA probe with a specific activity of 6-8.10(6) cpm/microgram DNA. In situ hybridization is as efficient on paraffin sections as on cryostat sections for detecting albumin mRNAs. Perfusion fixation with a 4% paraformaldehyde solution results in homogeneous RNA retention within tissue blocks, in contrast with immersion fixation, which yields heterogeneous RNA preservation. Comparison of immersion fixation with three different fixatives (paraformaldehyde, ethanol-acetic acid, and Bouin's fixative) shows that the highest level of hybridization signal is obtained with paraformaldehyde. Ethanol-acetic acid and Bouin's fixative appear less efficient for albumin mRNA detection. Loss of mRNAs within liver tissue blocks over time is largely although not completely prevented by paraffin embedding.     

Effect of fixation on immunolocalization of transferrin and albumin in the liver of the adult rat

The influence of the fixation procedure on the localization of albumin and transferrin in adult rat liver has been carried out using an indirect immunoperoxidase technique at the light and electron microscopic levels. Perfusion and immersion fixations with different concentrations of paraformaldehyde (with or without addition of glutaraldehyde) have been investigated. According to the mode of fixation (perfusion versus immersion) and the concentration of the fixative, the number of albumin and transferrin containing hepatocytes could vary from 10% to 100%, and different labeling patterns could be observed at the electron microscopic level. For the same concentration of fixative, a perfusion fixation induces a less intense labeling than an immersion fixation. Thus similar results are obtained after immersion fixation in 6% paraformaldehyde + 0.25% glutaraldehyde or after perfusion fixation in 4% paraformaldehyde + 0.025% glutaraldehyde. Similar data are noticed after immersion fixation in 4% paraformaldehyde or after perfusion fixation in 1% paraformaldehyde + 0.025% glutaraldehyde. Moreover, perfusion fixation induced a more fine cell structure preservation than immersion fixations and avoided the appearance of zones of fixation.     

Immunohistochemical localization of albumin and in situ hybridization of albumin mRNA

Hepatocytes actively involved in albumin synthesis were identified by immunohistochemical method. In sections of perioidate-lysine-2 per cent (w/v) paraformaldehyde fixed normal rat liver, albumin was detected in all hepatocytes. At the ultrastructural level, albumin was localized in the rough endoplasmic reticulum and in Golgi complexes located near the nucleus in only a small subpopulation of hepatocytes, while all other hepatocytes contained albumin only in Golgi complexes located near the bile canaliculi. Stimulation of albumin synthesis by puromycin aminonucleoside-induced nephrosis resulted in an altered intracellular distribution of albumin at the light microscopic level. When examined at the ultrastructural level, albumin was localized in the rough endoplasmic reticulum as well as in Golgi complexes located near the nucleus in nearly all these hepatocytes. Hepatocytes with the potential to synthesize albumin were identified by in situ hybridization of albumin mRNA. In sections of 0.1 per cent (v/v) glutaraldehyde perfusion fixed normal rat liver, albumin mRNA was detected in the cytoplasm of only a few hepatocytes scattered throughout the lobule. Following stimulation of albumin synthesis by the induction of nephrosis, albumin mRNA was detected in the cytoplasm of the hepatocytes. The source of albumin in those hepatocytes which lacked albumin mRNA was identified in analbuminemic rats injected with rat albumin. At 6 h post injection, the light microscopic distribution of albumin in the liver of these animals was virtually indistinguishable from that in normal rat liver. At the ultrastructural level, injected albumin was localized in lysosomes and in Golgi complexes located near the bile canaliculi.     

一种改良的肌细胞骨架染色方法

为了观察肌细胞骨架,对传统考马斯亮蓝染色法进行改良,并与免疫荧光染色法进行了比较。培养的血管平滑肌细胞先用多聚甲醛预固定后再进行考马斯亮蓝染色,可使细胞骨架非常清晰的显色,解决了传统考马斯亮蓝染色易使肌细胞变形、脱片的问题,其效果与免疫荧光染色相近。因此,多聚甲醛预固定．考马斯亮蓝染色法是一种适于肌细胞骨架染色的简便方法。     

AN ARTEFACT IN RADIOAUTOGRAPHY DUE TO BINDING OF FREE AMINO ACIDS TO TISSUES BY FIXATIVES

The binding of labeled free amino acids to liver and to purified protein by commonly used fixatives was investigated. Glutaraldehyde caused 25% of free leucine to be bound to serum albumin in solution, whereas formaldehyde bound only 0.5%. Liver slices were incubated for 2 min in the presence of labeled leucine and of puromycin, which permits absorption of leucine into the cell but inhibits incorporation into protein. Both counting and radioautographic techniques showed that glutaraldehyde bound 30 times, and osmic acid six times, as much free amino acid as did formaldehyde. By comparing liver slices incubated with and without puromycin for 2 min, it was calculated that in radioautographs prepared after fixation with glutaraldehyde, osmic acid, or formaldehyde 63, 25, and 4% respectively of the grains were due to binding of free amino acid. Formaldehyde, freshly prepared from paraformaldehyde, gives good preservation and is the recommended fixative for radioautography. When levels of free substrate in a tissue are high at the time fixative is added, the amount of binding of free substrate induced by the fixative should be included as a control in radioautographic experiments.     

Influence of cell fixatives and incubation schedule on PAP labeling of fibronectin in cultivated fibroblasts

A simple combined method for differential PAP labeling of fibronectin (FN) in mouse embryo fibroblast cultures was developed. Methanol-5% glacial acetic acid in dry ice-fixed cell monolayers showed mainly intracellular FN staining. Fixation with neutral paraformaldehyde before labeling, developed membrane- and extracellular matrix-associated FN. A combination of both procedures, which required incubation with primary antibody, fixation with paraformaldehyde followed by chilled acid methanol, and re-incubation with primary antibody, yielded sharp intracellular and extracellular FN labeling. The outlined methods can be readily employed in association with other staining techniques.     

Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins.

A cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intracellular proteins. Paraformaldehyde/methanol-fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cells fixed with paraformaldehyde or methanol alone (p less than 0.002) and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde (p less than 0.006). With paraformaldehyde/methanol fixation, cell morphology was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti-leukocyte antibodies was unaffected by fixation with paraformaldehyde/methanol. Paraformaldehyde effects on the intensity of DNA staining with propidium iodide were dependent on paraformaldehyde concentration and fixation temperature; these effects were least pronounced at low paraformaldehyde concentrations (0.25% or less), and at temperatures lower than 37 degrees C. Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peaks in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation-induced effect is useful in identifying biologically distinct near-diploid subpopulations in tumors.     

Analytical electron microscopic evaluation of the effects of paraformaldehyde pretreatment on the reaction of glutaraldehyde with biogenic amines

Analytical electron microscopy was used to determine the quantitative effects of paraformaldehyde pretreatment on the formation of the biogenic amine-glutaraldehyde-chrome complex. Pretreatment with paraformaldehyde prevented the glutaraldehyde-chrome reaction with norepinephrine in the rat adrenal medulla. In contrast to the effect of paraformaldehyde on norepinephrine, pretreatment did not prevent the chrome reaction in serotonin-containing argentaffin cells of the gut. X-Ray energy spectrographic analysis revealed a significant decrease in chrome content in the paraformaldehyde treated tissue, but sufficient chrome did react to produce an electron-dense product. Thus by treating tissue with paraformaldehyde prior to the glutaraldehyde chrome procedure, serotonergic sites may be differentiated from catecholaminergic areas at the electron microscopic level.     

Adverse Effect of Ammonium Salts on the Antibacterial Activity of Paraformaldehyde Solutions

The antibacterial activity of aqueous solutions of paraformaldehyde in concentrations from 0.1 to 0.4% (w/v) is bacteriostatic rather than bactericidal in the presence or absence of ammonium chloride. The presence of ammonium chloride significantly lengthened the time of exposure to paraformaldehyde necessary for inhibition of growth of the test organism (Staphylococcus aureus FDA 209) when unbuffered solutions were used. Elevation of the pH of the reacting mixture of paraformaldehyde and ammonium chloride by partial buffering lengthened the time of exposure necessary for inhibition of growth of the test organism. Decrease of antibacterial activity was concomitant with the disappearance of paraformaldehyde from the reacting mixture. The reaction of paraformaldehyde with ammonium chloride was rapid at room temperature (25 C) and at pH levels near neutrality. The fate of the reacting paraformaldehyde, including the possibility of the formation of hexamethylenetetramine or methylenimine, is discussed with particular reference to loss of antibacterial activity.     

Biosynthesis of Rat serum albumin

1. The labelling of intracellular and extracellular serum albumin was studied in liver slices and in whole rats by using new methods for the purification of the protein. 2. The results suggest that a polypeptide precursor is formed that is converted relatively slowly into serum albumin. 3. The effect of liver cell K(+) has been examined by a double-label method and it is shown that K(+) accelerates the rate of conversion of ;precursor' into albumin. The rate of transit of albumin across the cell membrane appears to be unrelated to the concentration of K(+) within the cell. 4. The time-course of incorporation of radioactive amino acid into albumin follows a sigmoidal mode. There is a pronounced time-lag before label starts to appear in intracellular albumin, and a further time-lag before it appears in extracellular albumin. 5. In slices the sum of intra- and extra-cellular label rises steadily from 30min after the start of labelling with a pulse of labelled leucine or valine and continues to rise for at least another 60min. This occurs whether labelling is stopped by addition of excess of carrier amino acid or with cycloheximide (100mum) or both. 6. The intracellular albumin content remains constant whether slices are maintained with low or normal intracellular K(+) concentrations. 7. Specific radioactivities of intracellular albumin (and fractions thereof) and of extracellular albumin were determined in vitro and in vivo. The results show that the intracellular albumin cannot be a precursor of extracellular albumin, unless a very small compartment is turning over much more rapidly than the bulk of the liver albumin or even of the microsomal albumin.     

Effect of Relative Humidity on the Penetrability and Sporicidal Activity of Formaldehyde

The effect of relative humidity (RH) on formaldehyde penetration of paper, glassine, and cotton was determined by the death rate of bacterial spores in glass tubes covered with these materials. The data show that paper is readily penetrated regardless of RH, but the RH greatly affects the penetration rate of glassine and cotton. A comparison was also made of the effect of RH on the penetrability of formaldehyde generated from Formalin and paraformaldehyde. At low RH, all three closures were penetrated more readily by formaldehyde from paraformaldehyde than from Formalin, but no difference in the two was observed at high RH. It is felt that the difference at low RH is primarily due to the condensation of the vaporized Formalin.     

New freeze-dry and vapor fixation method for immunohistochemistry of soluble proteins: subcellular location of the progesterone receptor

We describe a new application of freeze-drying and vapor fixation for immunohistochemical location of soluble proteins. The method avoids the liquid phase, which eliminates the possible diffusion of soluble proteins. Two vapor fixatives, paraformaldehyde and p-benzoquinone, were tested and p-benzoquinone was found to preserve antigenicity of progesterone receptor (PR) and ovalbumin better than paraformaldehyde. The method proved to be highly sensitive, since higher concentrations of antigen were found in some tissues and some tissues found to be antigen negative by earlier liquid fixation methods proved to contain antigen. The location of PR as a highly soluble protein was studied. With the present method, both unoccupied and occupied PR were located in the nuclei, a similar finding as with the earlier liquid fixation method. The results further support the concept that PR is an intranuclear protein independent of its ligand occupation. PR was detected in a few cells inside the follicles of the bursa of Fabricius and in the smooth muscle cell nuclei of the small intestine, observations not previously made owing to the insensitivity of the earlier methods.     

Formaldehyde prepared from paraformaldehyde is stable

For critical histological investigations, tissue fixation is sometimes carried out in formaldehyde freshly prepared from paraformaldehyde by heating. The purity of formaldehyde produced in this way is superior to that of commercial stock solutions. We studied the stability of freshly prepared formaldehyde solutions by determination of pH and titration of acid, which reflect the formation of formic acid. It was found that very small amounts of acid are produced during the heating of paraformaldehyde. Prolonged heating or storage of freshly prepared formaldehyde for up to 8 days did not significantly increase the amount of acid. It was also found that heating of the paraformaldehyde is not necessary, since depolymerization may take place at room temperature.

We conclude that formaldehyde prepared from paraformaldehyde remains stable for considerable periods of time, and it is therefore unnecessary to prepare it immediately prior to fixation. Also, in many cases, buffering of the fixative may be omitted, since only minor changes in the pH occur during fixation.     

Fixation and decalcification of adult zebrafish for histological, immunocytochemical, and genotypic analysis

To facilitate the molecular analysis of tissues in adult zebrafish, we tested eight different fixation and decalcification conditions for the ability to yield DNA suitable for PCR and tissue immunoreactivity, following paraffin embedding and sectioning. Although all conditions resulted in good tissue histology and immunocytochemistry, only two conditions left the DNA intact as seen by PCR. The results indicate that zebrafish fixed in either 10% neutral buffered formalin or 4% paraformaldehyde, followed by decalcification in 0.5 M EDTA, is an easy and reliable method that allows molecular experiments and histology to be performed on the same specimen. The fixation and decalcification by Dietrich's solution permitted the PCR amplification of DNA fragments of 250 but not 1000 bp. Therefore, a protocol of formalin or paraformaldehyde fixation followed by decalcification with EDTA is broadly applicable to a variety of vertebrate tissues when excellent histological, immunocytochemical, and genotypic analyses may be simultaneously required.     

Formaldehyde prepared from paraformaldehyde is stable

For critical histological investigations, tissue fixation is sometimes carried out in formaldehyde freshly prepared from paraformaldehyde by heating. The purity of formaldehyde produced in this way is superior to that of commercial stock solutions. We studied the stability of freshly prepared formaldehyde solutions by determination of pH and titration of acid, which reflect the formation of formic acid. It was found that very small amounts of acid are produced during the heating of paraformaldehyde. Prolonged heating or storage of freshly prepared formaldehyde for up to 8 days did not significantly increase the amount of acid. It was also found that heating of the paraformaldehyde is not necessary, since depolymerization may take place at room temperature.

We conclude that formaldehyde prepared from paraformaldehyde remains stable for considerable periods of time, and it is therefore unnecessary to prepare it immediately prior to fixation. Also, in many cases, buffering of the fixative may be omitted, since only minor changes in the pH occur during fixation.     

多聚甲醛固定对流式细胞术检测HepG2细胞整合素αVβ3的影响

目的:检测HepG2细胞表面整合素αVβ3的表达情况,同时探讨多聚甲醛的固定处理对HepG2细胞表面αVβ3检测的影响。方法:分别用0.25%的胰蛋白酶和1%的EDTA消化收集HepG2细胞,以FITC标记的抗αVβ3单抗检测细胞表面αVβ3的表达情况;在检测中,用2%浓度的多聚甲醛固定细胞后,采用流式细胞仪测定多聚甲醛固定组和未固定组HepG2细胞的平均荧光强度和阳性细胞率,对结果进行统计学分析。结果:以胰蛋白酶消化HepG2细胞后,抗αVβ3单抗标记的阳性细胞率为3.6%,远低于EDTA消化组的阳性细胞率(47%)。多聚甲醛固定组的平均荧光强度为2.23,阳性细胞率为4.6%;而未固定组的平均荧光强度为6.97,阳性细胞率为30.1%。以上结果经统计学分析,均具有显著性差异(P<0.05)。结论:HepG2细胞表达整合素αVβ3,多聚甲醛固定处理可干扰对HepG2细胞αVβ3的检测。     

多聚甲醛固定对荧光蛋白分子间FRET效率的影响

目的：研究多聚甲醛固定对利用荧光共振能量转移(fluorescence resonance energy transfer, FRET)检测细胞中蛋白质相互作用的影响,解决运动能力较强的细胞中FRET效率检测的问题。方法：选用两个已知能够相互作用的蛋白分子TRA和TRB,将荧光蛋白ECFP和EYFP的编码基因通过融合PCR分别标记在其C端;将两个融合基因共转染靶细胞,一组细胞经低浓度（0.5%）多聚甲醛短时（0.5~1h）固定,另一组不固定,利用激光共聚焦扫描显微镜检测两个融合蛋白之间的FRET效率,比较其在两组细胞之间的差异情况。结果：经过统计学分析,在活细胞和经低浓度多聚甲醛短时间固定的细胞中,ECFP与EYFP之间的FRET效率没有显著差异。结论：低浓度短时间的多聚甲醛固定对于荧光蛋白分子之间的相互作用没有显著的影响,因此对于运动能力过强的细胞可以固定后再进行FRET检测。     

Identification and characterization of membrane estrogen receptor from MCF7 estrogen-target cells

Estrogens control the proliferation of estrogen-target cells through a receptor mediated pathway. We have recently presented evidence that estradiol cancels the proliferative inhibition exerted by albumin on estrogen-target cells (indirect-negative hypothesis). We postulate that this mechanism requires the presence of a membrane estrogen receptor (mER)-membrane albumin receptor complex. Confirmation for mERalpha in MCF7 cells is now made using both the C542 monoclonal and ER-21 polyclonal antibodies (Ab)s specific for ERalpha. Western blot analysis of purified membrane proteins with ERalpha Abs revealed multiple high M(r) mERs (92 k, 110 k, and 130 k M(r)), as well as a 67 k M(r) mER; immunoreactive proteins were competed by inclusion of 500-fold molar excess C542 peptide. Ligand blot analysis of similar extracts with estradiol-peroxidase identified several potential mERs as well; two of these proteins were also recognized by C542 and ER-21 Abs (110 and 67 k M(r)). Fluorescence, confocal and electron microscopy of MCF7 cells fixed in 2.0% paraformaldehyde/0.1% glutaraldehyde identified specific mERalpha sites by immunocytochemistry. Specific binding of 3H-17beta-estradiol was reduced by a 200-fold molar excess of unlabeled 17beta-estradiol, but not by testosterone and progesterone. These results suggest that the ER on the plasma membrane of MCF7 cells is similar, but not identical to its intracellular counterpart. We propose that the observed mER actively participates in the estrogen-mediated proliferation of MCF7 cells.     

Characterization of a plasma membrane-resident albumin-binding protein associated with the proliferation of estrogen-target,serum-sensitive cells

Estrogens control the proliferation of their target cells through a receptor-mediated pathway. Recently presented evidence suggests that estradiol cancels the proliferative inhibition exerted by human albumin (HA) and recombinant human albumin (rHA) on estrogen-target serum-sensitive cells (indirect-negative hypothesis). We postulate that this mechanism requires the presence of a plasma membrane estrogen receptor (mER) and a plasma membrane albumin-binding protein (mABP). Direct evidence confirming the presence of mERalpha in MCF7 cells has recently been presented. Herein, we now show that Western blot analysis of purified T47D membrane proteins with the C542 ERalpha specific monoclonal antibody also revealed specific, multiple M(r) mERs (67, 110, and 130k M(r)). In addition, Western blot analysis with an ABP antiserum revealed a potential 60k M(r) ABP in both MCF7 and T47D plasma membrane extracts. No such evidence was observed in similar extracts from ER-negative, serum-insensitive MDA-MB231 cells. Ligand blot analysis of similar plasma membrane extracts with bovine serum albumin confirmed the presence of a 60k M(r) ABP in MCF7 and T47D cells; again, no such evidence was observed in comparable extracts from MDA-MB231 cells. Fluorescence and confocal microscopy of MCF7 cells fixed in 2.0% paraformaldehyde/0.1% glutaraldehyde identified specific membrane ABP antigenic sites by immunocytochemistry. Serum-insensitive MDA-MB231 cells fixed and labeled similarly did not exhibit this mABP. These results suggest that the proposed mABP is expressed only in serum-sensitive estrogen-target cells and is not expressed in cells insensitive to the proliferative inhibition of HA and rHA. Also, the present data suggest that the proposed mABP may be the recognition mechanism by which both HA and rHA inhibit MCF7 and T47D cell proliferation.     

Ultrastructural identification of argyrophil and argentaffin cells of the gastric mucosa in the rat

Ultrastructure of endocrine cells impregnated in the rat gastric mucosa by Grimelius method (identification of argyrophilia) and by Masson--Hamperl method (identification of argentaffinity) and influence of various fixatives on the structure and properties of the secretory granules in these cells have been studied. Fixation of the material in paraformaldehyde or glutaraldehyde varies in its effect on the granule structure of EC-, D1- and ECL-cells, while its influence on the granule structure of G-, D- and AL-cells is identical. The granules of EC-, ECL- and G-cells are argyrophil, and only those of EC-cells are argentaffin. Weak argyrophilia, which is evidently not appearant at the light-optical level, is specific for granules of D1- and AL-cells. Fixation in paraformaldehyde and especially the subsequent treatment in osmium tetroxide results in increasing argyrophilia of the endocrine cells, as compared to fixation in glutaraldehyde. Varieties in the effect of the fixatives do not prevent ultrastructural and histochemical identification of the endocrine cell types.