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PubMed1996

p75 nerve growth factor receptor staining helps identify desmoplastic and neurotropic melanoma.

Melanoma is a malignant tumor with a varied histologic appearance. Melanoma composed of spindle cells may include desmoplastic and neurotropic melanoma. The histologic diagnosis of desmoplastic and neurotropic melanoma can be difficult. Although S100 protein stains a majority of these melanomas, the staining may be weak or focal. HMB-45, a more specific marker of melanoma, is frequently negative in desmoplastic and neurotropic melanoma. In order to aid the identification of desmoplastic and neurotropic melanoma, we stained 13 spindle cell melanomas (5 neurotropic melanomas, 5 desmoplastic melanomas, 3 spindle cell melanomas without either desmoplasia or neurotropism) with p75 NGF-R and compared the staining results with S100 and HMB-45. p75 NGF-R is the low affinity nerve growth factor receptor reported to be present on the surface of neural-crest-derived cells. Conventional melanoma as well as neurotized nevi, neurofibroma, spindle squamous carcinoma, atypical fibroxanthoma, dermatofibroma and scars were also stained with p75 NGF-R. p75 NGF-R stained all of the desmoplastic and neurotropic melanomas tested. In each of these cases, negative HMB-45 staining of the spindle cells was seen. In many cases the number and intensity of the spindle cells staining with p75 NGF-R was greater than with S100. Neurofibroma, neurotized nevi and focal cells in round cell melanoma also were stained with p75 NGF-R. All the squamous cell carcinomas, atypical fibroxanthomas, dermatofibromas and scars were negative for p75 NGF-R. Based on our results, p75 NGF-R may be useful as an additional confirmatory antibody in a melanoma panel, especially in differentiating desmoplastic and neurotropic melanomas from non-neural-crest-derived spindle cell lesions. We feel it also can be helpful in better identifying margins of excision of these melanomas. p75 NGF-R, like S100 protein, will not differentiate desmoplastic and neurotropic melanomas from other neural-crest-derived lesions.

Antigens, Neoplasm↗

PubMed1985

Staining patterns of human pre-malignant oral epithelium and squamous cell carcinomas by monoclonal anti-keratin antibodies.

Formalin-fixed, paraffin-embedded biopsies of metaplastic keratinized oral mucosa (fibromas and leukoplakias), oral mucosa with epithelial dysplasia and oral squamous cell carcinomas were stained with two monoclonal anti-keratin antibodies (AE1 and AE2). Intense suprabasal staining was seen with AE1 in metaplastic keratinized epithelium, whereas staining of adjacent normal unkeratinized epithelium generally was restricted to basal cells. In dysplastic epithelium and squamous cell carcinomas, staining with AE1 revealed a highly disturbed anti-keratin staining pattern. AE2 stained metaplastic keratinized epithelium in a suprabasal pattern but adjacent unkeratinized epithelium did not stain. In dysplastic epithelium and squamous cell carcinomas, AE2 staining was variable and sometimes absent. Further studies are indicated to clarify whether changes in anti-keratin staining patterns can be used for diagnostic and prognostic purposes.

Antibodies, Monoclonal↗

PubMed1987

Reversible and reproducible in vivo staining of motor endplates by use of the acetylthiocholine-iodide method.

In vivo staining of motor endplates was carried out in the anterior tibial muscle of the rat, using the acetylthiocholine-iodide staining method of Koelle and Friedenwald. A strong and distinct endplate staining was obtained after incubation in the staining solution for only one minute if the epimysium had been removed from the muscle. Staining with intact epimysium gave a less satisfactory result if the incubation time was 3 minutes, but it was excellent after incubation for 10 minutes. At re-examination 5 or 30 days later, the initial staining had disappeared in all animals, and a second staining at this time gave just as good results as on the first occasion. The inflammatory reaction in the muscle was more severe with removed epimysium than with intact epimysium, and the staining solution had a slight local toxic effect compared with 0.9% NaCl. Staining with intact epimysium should therefore be preferred if possible.

Acetylcholinesterase↗

PubMed1987

A new method to determine Giardia cyst viability: correlation of fluorescein diacetate and propidium iodide staining with animal infectivity.

The viability of Giardia muris cysts was studied with the fluorogenic dyes fluorescein diacetate (FDA) and propidium iodide (PI). G. muris cysts were seen to fluoresce intensely green with FDA at an excitation wavelength of 450 to 490 nm. Cysts stained with PI fluoresced bright orange at an excitation wavelength of 450 to 490 nm and bright red at 545 to 546 nm. Examination of isolated G. muris cyst preparations stained with FDA-PI revealed that greater than 85% of the cysts stained green with FDA and less than 15% stained orange-red with PI. Using the mouse model for giardiasis, we inoculated FDA- or PI-stained cysts into neonatal mice. Feces were examined at days 3, 5, 8, and 11 postinoculation for the presence of cysts. Using 1,000 FDA-stained cysts as the inoculum, we detected cysts at days 5, 8, and 11 postinoculation in 19 of 19 mice, whereas a 50-fold greater dose of cysts produced infection in 27 of 27 mice at day 3 as well as at days 5, 8, and 11 postinoculation. Inoculation of mice with either 5,000 or 50,000 PI-stained G. muris cysts did not produce infection in any of the animals. Necropsy of mice infected with FDA-stained cysts showed trophozoites within the intestines. No trophozoites were detected within animals inoculated with PI-stained cysts. These results demonstrate that FDA-positive cysts are viable, as determined by infectivity, while PI-positive cysts are nonviable and incapable of producing G. muris infections in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1998

Evaluation of intestinal protozoan morphology in human fecal specimens preserved in EcoFix: comparison of Wheatley's trichrome stain and EcoStain.

As a result of disposal problems related to the use of mercury compounds, many laboratories have switched from mercuric chloride-based Schaudinn's and polyvinyl alcohol (PVA) stool preservatives to other, non-mercury-based preservatives. A comparison of organism recoveries and morphologies of the intestinal protozoa was undertaken with PVA containing the EcoFix zinc-based Schaudinn's preservative (Meridian Diagnostics, Inc.); both Wheatley's modification of Gomori's trichrome stain (WT) and EcoStain (ES) were used to stain 51 human fecal specimens. Morphology, clarity of nuclear and cytoplasmic detail, overall color differences, and the ease or difficulty in detecting intestinal protozoa in fecal debris were assessed for the two permanent stained smears. Overall, organism morphology of the intestinal protozoa stained with WT and that of protozoa stained with ES were not equal in nuclear and cytoplasmic detail or range of color. However, the same organisms were identified in stained fecal smears with either WT or ES, with the exception of situations in which organism numbers were characterized as rare. Included were 67 protozoan challenges (number of organisms): Entamoeba histolytica-Entamoeba dispar (5), Entamoeba coli (9), Entamoeba hartmanni (6), Endolimax nana (12), Iodamoeba bütschlii (8), Blastocystis hominis (19), Giardia lamblia (6), Dientamoeba fragilis (2), yeast (2), and leukocytes (2). Five specimens were negative for parasites but contained fecal debris that was compared for morphologic detail and color range. The ES produces a more gray-green monotone with very little pink or red tone; contrast among the various colors is less than that seen with WT. Stain intensity for all organisms was acceptable, and there were no problems with stain deposition. The quality of the protozoan morphology with ES was often comparable to that with WT (36 of 67 [53.7%]) and, in some cases, better (24 of 67 [35.8%]). Organisms on the WT-stained smear exhibited better morphology in a few instances (4 of 67 [6%]), and in three instances, there were discrepant organism numbers.

Animals↗

PubMed1978

Comparison of immunofluorescence and immunoperoxidase staining for identification of rubella virus isolates.

To explore possible advantages which immunoperoxidase (IP) staining might have over immunofluorescence (IF) staining for identifying rubella virus isolates, direct comparative studies were done on the same coded clinical materials using the same rubella immune rabbit serum as the primary antiserum in both systems. The rubella immune rabbit serum and conjugated anti-rabbit immune globulins could be used more dilute in the IP system than in the IF system. Both IP and IF staining detected rubella antigen in all specimens which were positive by interference. IP staining also detected low levels of rubella antigen in a few additional specimens which had originally been positive for rubella virus, but which on retesting were negative by interference and IF staining. With second-cell-culture-passage material, IP and IF staining showed comparable specificity, and the few specimens which reacted nonspecifically generally did so in both systems. Cell cultures inoculated directly with urine specimens showed greater nonspecificity by IP than by IF, but this activity could be abolished by pretreatment with sodium azide and peroxide; other methods tried for inactivating endogenous peroxidase activity destroyed rubella antigen as well. The intensity of staining for positive specimens was comparable in the two systems. However, more antigen was demonstrable in both systems when BHK-21 cells were inoculated as a cell suspension and then permitted to grow into monolayers than when the same specimens were inoculated into preformed monolayers. IP staining was considered to be a highly satisfactory alternative to IF staining for identification of rubella virus isolates.

Animals↗

PubMed2000

Wet mount microscopy reflects functional vaginal lactobacillary flora better than Gram stain.

AIM: The status of vaginal lacto-bacillary flora, an indicator of possible genital infection and pregnancy complications, can be assessed on wet mount or Gram stained specimens. The former is quick, the latter more routine. The accuracy of the two preparative techniques to detect normal vaginal lacto-bacillary microflora was compared for 646 patients. The effect of delay in transport medium before Gram staining was also investigated. METHODS: Patients presented with infectious vaginitis or for a routine prenatal visit. After placement of a speculum, duplicate smears were taken from the upper vaginal vault and examined fresh or after Gram staining. Lacto-bacillary grades from both methods were compared with lactate concentration in vaginal rinses. In a subgroup of 238 patients, Gram staining was performed both on fresh smears and those that had been transported in Stuart's growth medium. RESULTS: Higher lacto-bacillary grades (more disrupted flora) were diagnosed 2.9 times more frequently on Gram stained specimens than on wet mounts (p < 0.0001), a difference even more pronounced after transport in Stuart's medium (relative risk, 4.2; p < 0.0001). Lacto-bacillary grades assessed on wet mounts correlated better with vaginal lactate concentration than those assessed on Gram stains. CONCLUSIONS: Easier recognition of lacto-bacillary morphotypes on wet mounts than on Gram stains might result from the loss of lactobacilli by the process of fixation or Gram staining. Wet mount microscopy of vaginal smears for assessment of lacto-bacillary grades, rather than Gram staining, is strongly recommended.

Culture Media↗

PubMedDate not supplied

Inhibitors of the polymerase chain reaction in Papanicolaou stain. Removal with a simple destaining procedure.

OBJECTIVE: To identify inhibitors of the polymerase chain reaction (PCR) within Papanicolaou stain and to develop a method of removal. STUDY DESIGN: PCR was performed on cellular digests of archival, Papanicolaou-stained cervical smears and unstained cervical smears using primers to the human beta-globin gene to determine if Papanicolaou stain inhibited the reaction. Next, in order to identify the specific reagents responsible for inhibition, components of the Papanicolaou stain were added to PCRs performed on purified human genomic DNA. Finally, PCR was performed on cellular digests of cervical smears that had been destained using 1% HCl to determine if the inhibitors of PCR were removed. RESULTS: Cellular digests from Papanicolaou-stained cervical smears did not yield products from PCR, whereas cellular digests from unstained cervical smears always yielded PCR products. Analysis of individual Papanicolaou stain reagents revealed inhibition of PCR by hematoxylin and aluminum sulfate. These inhibitors could be removed from Papanicolaou-stained cervical smears by destaining the slides with 1% HCl. CONCLUSION: The hematoxylin and aluminum sulfate within Papanicolaou stain inhibit PCR but can be removed from Papanicolaou-stained cytologic specimens using a simple destaining procedure.

Alum Compounds↗

PubMed1987

Value of in vivo mucosa-staining test with methylene blue in the diagnosis of pretumoral and tumoral lesions of the bladder.

An in vivo staining test, methylene blue was applied to 50 patients with bladder tumor. 378 biopsies were performed in 85 areas stained with methylene blue and in 293 random biopsy areas. Of these samples, 56 had carcinoma in situ: 38 of these were not stained (68% false-negative) and the remaining 18 took up the stain. In the latter corresponding to 11 patients, carcinoma in situ was also shown in unstained areas examined by random biopsies. In 37 areas with urothelial dysplasia, 31 were not stained (84% false-negatives) while 6 cases (16%) took up the stain. Bladder tumors were stained in an irregular way but the intensity of the stain was related to the tumor grade. According to our experience the in vivo staining test with methylene blue cannot be used as a marker for pretumoral and tumoral lesions of the bladder.

Adult↗

PubMed1997

Differential staining of apoptotic nuclei in living cells: application to macronuclear elimination in Tetrahymena.

Acridine orange (AO) has been used as a vital fluorescent stain to identify apoptotic cells in Drosophila, but little is known about what structures are stained. We explored the specificity of AO staining while studying nuclear apoptosis in Tetrahymena. Using AO alone or together with the vital nuclear stain Hoechst 33342 (HO), we find that lysosomes are generally clustered around the degenerating nucleus and that such nuclei are stained an orange-red color, like lysosomes. Significantly, the combined dyes, more so than with AO alone, distinguish between apoptotic and normal (or necrotic) nuclei by a clear color difference. Moreover, these dyes differentially stain apoptotic and normal nuclei in avian chondrocytes. The differential staining results are nullified in fixed cells or in cytoskeletal preparations treated with RNAse. Similarly, lysosomotrophic agents eliminate the differential staining. Our results are consistent with acidification of the apoptotic nucleus, possibly by fusion with lysosomes. However, even under basic conditions, the macronucleus condenses and is eliminated, suggesting that, if the nucleus is becoming acidified, acidification by itself is not essential for nuclear elimination. The differential staining procedure may provide a useful method for specifically identifying apoptotic cells and separating them for further analysis.

Acridine Orange↗

PubMed1997

Patterns of hyaluronan staining are modified by fixation techniques.

The apparent intensity of hyaluronan (HA) staining in tissue sections can vary as a function of fixation techniques. We examined the histochemical distribution of HA in normal human skin using an HA-specific binding peptide derived from bovine nasal cartilage. The HA, particularly in the dermis, was best preserved in sections fixed in 10% acid-formalin with 70% ethanol. In contrast, sections fixed in the routine 10% neutral-buffered formalin had a much weaker intensity of HA staining. Furthermore, acid-formalin/ethanol-fixed sections retained much of their apparent HA after incubation with saline, in contrast to the neutral formalin-fixed sections, in which most of the stainable HA was lost. Such marked differences in staining intensity were not observed in slides stained with Alcian blue, a procedure presumed to stain HA as well as other glycosaminoglycans. Staining using the HA binding peptide was entirely absent when sections were first preincubated in hyaluronidase, whereas similar Alcian blue-stained sections retained most of their staining intensity. Caution should be exercised in evaluating the distribution of HA in tissues using the HA binding peptide, particularly when different fixation techniques among several laboratories are being compared. In addition, the ability to evaluate the HA content of tissues using Alcian blue staining should be reconsidered. The sulfated glycosaminolglycans of the "ground substance" appear to be the predominant substrates for Alcian blue.

Adult↗

PubMed1992

Procedures for specific detection of silver-stained nucleolar proteins on western blots.

Nucleolar organizer region (NOR)-silver staining of the chromosomes and nucleoli is a method that enables the detection of proteins associated with the ribosomal genes. We adapted the most commonly used cytochemical NOR-silver staining techniques to Western-blotted proteins of HeLa cells, mimicking the silver staining of cells in situ, and testing several parameters that may influence the in situ reaction. Two of these techniques, both one-step methods with colloidal developers, were standardized to obtain reproducible results. The specificity of NOR staining is documented by: (a) only a few bands are revealed among the many proteins detected by total proteins staining on gels or blots; two major groups of bands are found around 100 KD and 40 KD that could correspond at least in part to nucleolin and B23 nucleolar proteins; (b) the silver staining of bands was not the result of the high relative protein concentrations; and (c) the same number of NOR-silver-stained bands was observed across a large range of protein concentrations. The reaction appeared to be specific for a subset of nucleolar proteins, because the same bands were observed with the use of nucleolar, nuclear, or total cell protein extracts, and the silver grains observed in electron microscopy were clearly confined to the nucleolar fibrillar centers and dense fibrillar component. The efficiency of the reaction was not modified by any of the tested fixative pre-treatments except that involving methanol. The presented standardization of NOR-silver staining on Western blots allows the characterization of the Ag-NOR proteins and their specific regions responsible for silver staining of the nucleolus.

Blotting, Western↗

PubMed2006

Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA.

BACKGROUND: Gene expression profiling by microarray analysis of cells enriched by laser capture microdissection (LCM) faces several technical challenges. Frozen sections yield higher quality RNA than paraffin-imbedded sections, but even with frozen sections, the staining methods used for histological identification of cells of interest could still damage the mRNA in the cells. To study the contribution of staining methods to degradation of results from gene expression profiling of LCM samples, we subjected pellets of the mouse plasma cell tumor cell line TEPC 1165 to direct RNA extraction and to parallel frozen sectioning for LCM and subsequent RNA extraction. We used microarray hybridization analysis to compare gene expression profiles of RNA from cell pellets with gene expression profiles of RNA from frozen sections that had been stained with hematoxylin and eosin (H&E), Nissl Stain (NS), and for immunofluorescence (IF) as well as with the plasma cell-revealing methyl green pyronin (MGP) stain. All RNAs were amplified with two rounds of T7-based in vitro transcription and analyzed by two-color expression analysis on 10-K cDNA microarrays. RESULTS: The MGP-stained samples showed the least introduction of mRNA loss, followed by H&E and immunofluorescence. Nissl staining was significantly more detrimental to gene expression profiles, presumably owing to an aqueous step in which RNA may have been damaged by endogenous or exogenous RNAases. CONCLUSION: RNA damage can occur during the staining steps preparatory to laser capture microdissection, with the consequence of loss of representation of certain genes in microarray hybridization analysis. Inclusion of RNAase inhibitor in aqueous staining solutions appears to be important in protecting RNA from loss of gene transcripts.

Animals↗

PubMed2000

Orcein-picroindigocarmine--a new multiple stain.

A new "orcein-picroindigocarmine staining", a colour combination of orcein, indigo carmine, and picric acid, was developed for histological applications. The new technique was tested on different human tissues. Colours ranging from red to brown, yellow, green and blue were observed in paraffine sections of tissues stained by this method. Nuclear structures in all tissues were stained dark brown to dark blue. Squamous epithelium was stained light brown with varying shades of blue in upper horny layers, whereas the ciliated epithelium was tinged blue grey. When connective tissue was stained, collagen fibrils appeared strongly blue next to elastic fibres, which took on a rust brown tinge; cellular components were all coloured brown. The matrix of hyaline cartilage was stained in different shades of blue, with the chondrocytes rust brown. Sections of bone components appeared dark blue to dark green. Skeletal muscle cells were coloured yellow and green with blue collagenous septa. The new staining is useful for distinguishing connective tissue components such as elastic fibres and collagen fibrils. It also demonstrates chondrocytes in favourable contrast to the cartilage matrix. The technique produces aesthetic staining colouring that could supplement histological investigations and provide an alternative to other staining materials.

Adult↗

PubMed1987

A quick, reliable staining technique for human sperm morphology.

The evaluation of sperm morphology is still an important parameter in the diagnosis of the infertile male. Most techniques used for staining human sperm are very time-consuming. A routine stain used for determining differential count of leucocytes (Diff-Quik stain) was evaluated against the standard Papanicolaou stain. Morphology results from 20 duplicate semen smears using both staining methods were determined separately by 2 technicians using a blind protocol. No significant differences were observed when comparing the two staining methods (paired Student's t-test). The advantages of the Diff-Quik stain are: a) complete staining-to-reading time under 7 min, b) commercially prepared reagents, and c) case of staining procedure.

Humans↗

PubMed1979

Selective vital staining of companion cells of potato tuber and parsnip root with neutral red.

When staining the internal phloem region of a potato tuber with the vital stain neutral red, it was observed that files of elongated cells of narrow diameter were heavily stained and were easily distinguishable from the more isodiametric parenchyma cells, many of which did not stain with neutral red. The elongated cells were identified as companion cells by locating the adjacent sieve-tube members through counterstaining with aniline blue and reviewing under violet light. Of a number of other plants surveyed, only parsnip roots possessed companion cells exhibiting a similar slective staining. In other plants both the companion cells and the surrounding parenchyma cells usually stained. Sieve-tube members never accumulated neutral red. It was concluded that the vacuoles of the companion cells of the potato tuber were stained by the ion trap mechanism because of the color of the accumulated stain, the lack of staining when neutral red was applied in an acidic solution, and the complete destaining after soaking in dilute ammonium hydroxide.

Histocytochemistry↗

PubMed1986

Detection of sperm histone diversity among vertebrates by alkaline fast green staining.

Testis sections from fifteen species from six classes of vertebrates were stained with alkaline fast green (AFG) to correlate staining differences with the known biochemical diversity of histones in the spermatozoa. After trichloroacetic acid (TCA) hydrolysis the sperm of some species known to contain sperm-specific histones did not stain. This correlation held if fixation in neutral buffered formalin was limited to 3 to 6 hr and hydrolysis was done at 90 C. The species whose sperm did stain after TCA hydrolysis could be divided into three groups. In some species the sperm no longer stained if, after TCA, the sections were treated with thioglycollic acid. These sperm contained basic proteins that were rich in cysteine. In turn, the group of species whose sperm continued to stain after TCA and thioglycollic acid treatments could be subdivided. The sperm of some were stained specifically without DNA hydrolysis if the AFG was made up with sodium chloride. These sperm contained sperm-specific histones. In other species the sperm did not stain under these conditions, and these sperm had a basic protein complement similar to that found in somatic cell nuclei. These correlations suggest that AFG staining can be used to detect sperm histone diversity in a wide range of organisms.

Animals↗

PubMed1987

The effect of pH on silver staining of nucleolus organizer regions.

The effect of pH on silver staining of the nucleolus organizer regions (NORs) of human chromosomes has been investigated between pH 6.5 and 12.0. Nonvolatile mixtures of ethanolamine and ethanolammonium nitrate replaced the ammonia of standard procedures. The optimal NOR staining obtained at pH 3.5 by the silver staining procedure of Howell and Black served as a standard; this procedure stained all ten NORs in 90% of mitoses. Similar NOR staining was found in 75% of mitoses stained at pH 11.7 or 11.8, but only in 10-15% of mitoses stained between pH 11.6 and 10.0. Between pH 10.0 and 9.0 NOR staining was incomplete, and between pH 8.5 and 6.5 there was no NOR staining.

Chromosome Banding↗