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PubMed2005

[Application of histochemical staining in diagnosis of osteosarcomas].

OBJECTIVE: To study the histochemical staining in the diagnosis of osteosarcoma. METHODS: To compare the effectiveness of picrosirius red, improved Ponceau trichrome and Masson trichrome staining methods on bone formation tissues in conventional osteosarcoma, paraosteal osteosarcoma, periosteal osteosarcoma, extraskeletal osteosarcoma, inflammatory myofibroblastic tumour, malignant fibrohistiocytoma, chondrosarcoma, fibrosis with ossification and calcification. RESULTS: With modified Ponceau trichrome staining, bone formation tissues showed a homogenous, orange-red interblended with blue in color. From osteoid to mature bone the color changed from orange-red, light blue to dark blue. Fibrotic tissue was stained blue in color with striated appearance. Cartilage was not stained. Picrosirius red method gave bone formation tissues homogenous staining. Along with bone maturation, from osteoid tissue to mineralized bones, the color showed changes from light red, yellow, orange-red, red to dark purple. The cartilage demonstrated homogenous light red in color. Fibrous tissue stained red interblended with yellow in color, striated in shape. With Masson trichrome staining osteoid displayed pale blue and mineralized bone showed dark blue in color. Fibrotic tissue showed a striated blue staining. CONCLUSION: The modified Ponceau trichrome and Picrosirius red staining methods are better than Masson trichrome to demonstrate bone formation tissue in osteosarcoma. The former two methods could be also used in study on bone formation.

Bone Neoplasms↗

PubMed2005

Performance of variations of carbolfuchsin staining of sputum smears for AFB under field conditions.

SETTING: A field project in Bangladesh. OBJECTIVE: To compare the effectiveness of commonly used carbolfuchsin staining variations. DESIGN: Routine hot Ziehl-Neelsen (ZN) 1% basic fuchsin staining for 15 min in 75 field clinics. Blind reading of duplicate smears stained by ZN 1% vs. 0.3% basic fuchsin applied for 5 min, or by ZN 1% 5 min vs. Kinyoun cold staining. Rechecking of discordant series. RESULTS: For comparable numbers of false positives, sensitivity was significantly lower with Kinyoun than with ZN 1% 5 min (85.6% vs. 93.0%, P < 0.001). Sensitivity with ZN 1% 5 min was not significantly higher than with 0.3% 5 min staining (89.9% vs. 86.5%). Routine examination using 1% 15 min ZN identified more positives than any of the study techniques. CONCLUSIONS: Kinyoun cold staining sensitivity was unsatisfactory in field clinics. The sensitivity of the WHO/IUATLD recommended 0.3% fuchsin for 5 min was not significantly different from the original 1% ZN for 5 min, but 1% 15 min hot staining might be superior. A reduced fuchsin concentration together with a short staining time may leave too narrow a margin for error. TB programmes using hot ZN with a concentrated stain or longer staining time should not be urged to change.

Bangladesh↗

PubMed1987

Scanning electron microscopy of black stain on human permanent teeth.

Black stain may develop on the coronal surfaces of human teeth, and this type of stain is common in the Hong Kong Chinese population. The present study was undertaken to ascertain if the deposit conforms in composition to the black stain found elsewhere, and to describe its ultrastructure using the scanning electron microscope. Gram-stained smears were made from black stain on the teeth of 11 adult Hong Kong Chinese and studied microscopically. From another 15 persons extracted teeth exhibiting black stain were obtained and fixed. Two ground sections were made from each tooth, one was stained with toluidine blue, the other was dried and prepared for scanning electron microscopy. The gram-stained smears demonstrated predominantly gram-positive filamentous microorganisms with an admixture of gram-positive cocci and rods. The ground sections revealed a deposit on the outer surface of the enamel, which was clearly divided into two distinctly different layers: an inner yellow opaque layer, and an outer layer of microorganisms. Scanning electron microscopy demonstrated that the deposit consisted entirely of microorganisms, and that in portions close to the enamel they were often obscured by a substance indicative of calcification. Thus the black stain found on the teeth of Hong Kong Chinese is similar in composition and structure to that reported to occur in other populations. The black stain is a special type of dental plaque characterized by its simple flora and its tendency to calcify.

Adult↗

PubMed1988

'Catalysts' for polyacrylamide gel polymerization and detection of proteins by silver staining.

The crosslinker diacrylyl-piperazine produces polyacrylamide gels which display improved electrophoretic separation of proteins and better physical strength. It also produces gels with improved detection of proteins by ammoniacal silver staining by reducing the background. This reduced background provided us with an opportunity to investigate residual background staining caused by the catalytic reagents utilized in the polymerization of acrylamide gels. The commonly used catalyst system, tetramethyl-ethylenediamine and ammonium persulfate was shown to be responsible for the yellow staining background found after a prolonged development time with silver staining. An alternate catalyst system has been designed to decrease further the formation of this background staining. Dimethyl-piperazine or tetramethylethylenediamine, potassium or ammonium persulfate, and sodium thiosulfate are shown to provide for gels which have excellent mechanical and staining characteristics. These catalytic systems produce little background staining despite prolonged development time with the ammoniacal silver stain, and they reduce background staining with the dichromate silver stain.

Acrylic Resins↗

PubMed1982

Basic dyes in the staining of DNA-phosphate groups and DNA-aldehyde molecules in cell nuclei.

A novel method is presented for the staining of cell nuclei with aqueous solutions of Janus blue, methylene blue, and Janus red in tissue sections from which RNA has been extracted selectively with cold phosphoric acid. Not only this, DNA-aldehyde molecules can also be stained when tissue sections from which RNA has been extracted are then hydrolysed in 6 N hydrochloric acid at 30 degrees C for 15 min followed by staining with Janus blue, methylene blue, and Janus red. Following staining with any of these dyes, sections can be dried between folds of filter paper and then treated with n-butanol or passed through grades of ethanol, cleared in xylene and mounted. Staining with Janus blue has been considered to be metachromatic, particularly in the sections of the rectum in which glycogen stains blue-black and the nuclei purplish. An aqueous solution of methylene blue does also stain glycogen blue with similar colour of the nuclei while with Janus red both the nuclei as well as glycogen stain red. The in situ absorption spectra of the nuclei stained with the dyes mentioned, after selective extraction of RNA, reveal peaks of maximum absorption at 560-570 nm (Janus blue), at 600 and 640 nm (methylene blue), and at 530 nm (Janus red). Those of nuclei stained for DNA-aldehyde molecules are at 560 nm (Janus blue), at 600 and 630 nm (methylene blue), and at 520 nm (Janus red). Possible significance of these findings has been discussed.

Aldehydes↗

PubMed1995

Decrease of heparan sulfate staining in the glomerular basement membrane in murine lupus nephritis.

Recently we found in biopsies of human lupus nephritis a nearly complete loss of heparan sulfate (HS) staining in the glomerular basement membrane (GMB). To clarify the relationship between HS staining and albuminuria in lupus nephritis, we studied MRL/lpr mice with short (< 7 days) or prolonged duration of albuminuria (14-21 days) and compared these with mice of different ages without albuminuria. Kidney sections were stained for mouse immunoglobulin (Ig), HS, heparan sulfate proteoglycan (HSPG)-core protein and laminin in immunofluorescence. In mice with prolonged albuminuria HS staining in the glomerular capillary loops had almost completely disappeared, whereas staining was unaltered in non-albuminuric mice (P = 0.001). In mice with short duration of albuminuria, there was a tendency toward a decrease of HS staining (P = 0.06). The expression of HSPG-core protein and other extra cellular matrix (ECM) components was unaltered in all groups. HS staining correlated inversely with albuminuria (rs = -0.55; P < 0.001) and with staining of Ig deposits in the capillary loops (rs = -0.74; P < 0.001). Despite the nearly complete loss of HS staining in the GBM in mice with prolonged albuminuria, there was no change in glomerular HS content as assessed by agarose electrophoresis and HS inhibition ELISA. We conclude that the development of albuminuria in MRL/lpr mice is accompanied by a loss of HS staining in the GBM, probably due to the masking of HS by deposits of Ig. In vitro studies revealed that autoantibodies complexed to nucleosomal antigens can inhibit the binding of the anti-HS monoclonal antibody to HS. Whether this also occurs in vivo remains to be determined.

Albuminuria↗

PubMed1993

New staining methods for semithin sections of tissues of Seriola dumerili embedded in methacrylate.

Semithin sections of different tissues of Seriola dumerili R. Embedded in glycol methacrylate, were stained with two staining methods normally used in conventional haematology and histology: Gutierrez's cytopanchrome and Gutierrez's polychrome V.O.F. The results observed in all tissues embedded in this resin and stained with V.O.F. are satisfactory and comparable, as to staining affinity, to those obtained on paraffin-embedded tissues. Gutierrez's trichrome stain appears to be a useful tool in the study of oogenesis for detecting changes in staining according to cellular structure and maturation stage. The results observed with cytopanchrome are similar to those obtained with toluidine blue in glycol methacrylate-embedded tissues. The use of these two staining techniques, requires no previous oxidation, alkali treatments, high temperatures or removal of the embedding medium. Furthermore, both dyes can be used at the same time: in this case, cytoplasm of previtellogenic oocytes showed a stronger affinity for cytopanchrome, and were more intensely stained by V.O.F. as the vitellogenesis of Seriola dumerili progressed. Both stain, and especially the cytopanchrome one, also are available for staining the liver, gills and testis.

Animals↗

PubMed1978

Demonstration of hepatitis B-surface antigen in liver biopsies. A comparative investigation of immunoperoxidase and orcein staining on identical sections of formalin fixed, paraffin embedded tissue.

Staining of 1000 consecutive liver biopsies with orcein showed positive reaction in the cytoplasm of ground-glass hepatocytes in 18 of the biopsies. On new sections of the 18 orcein positive biopsies immunoperoxidase staining was performed in order to demonstrate hepatitis B-surface antigen (HBsAg). After destaining the same sections were stained with orcein. All biopsies showing positive orcein staining showed positive immunoperoxidase reaction. The number of positive cells was in biopsies with less than 20 positive cells per mm2 biopsy larger using the immumoperoxidase staining than with orcein staining. Further the staining contrast was more pronounced. Immunoperoxidase staining thus seems more sensitive than orcein staining for demonstrating HBsAg in liver tissues. Orcein stains HBsAg in liver tissue even though the antigen determinants are blocked by antibodies.

Cytoplasm↗

PubMed1997

Cytokeratin 7 staining in mammary and extramammary Paget's disease.

There are a variety of routine and immunohistochemical stains used to diagnose mammary and extramammary Paget's disease (MPD and EMPD). Most of the stains commonly used, however, show a positive reaction in the Paget's cells in all cases. We wanted to assess which immunohistochemical stain is the best for the diagnosis of MPD and EMPD, as well as the best stain for identifying small foci of tumors in evaluating tumor margins. We evaluated nine cases of MPD and nine cases of EMPD, which were randomly chosen, with a battery of immunohistochemical stains. These stains included cytokeratin 7, cytokeratin 20, carcinoembryonic antigen, Ber-EP4, and CAM 5.2. Cytokeratin 7 was the only immunohistochemical stain that stained all of the cases diffusely, and, in all of the cases, the staining of the Paget's cell was intense and specific within the epidermis. We concluded that cytokeratin 7 is the immunohistochemical stain of choice in the diagnosis of Paget's disease. Because cytokeratin 7 seems to identify single cells, it might also be valuable in evaluating surgical margins for small foci in a tumor such as EMPD, which might have a multifocal origin.

Adult↗

PubMedDate not supplied

Evaluation of membranous staining of mesothelioma.

Panels of immunohistochemical markers are often used to aid in categorizing malignant neoplasms involving the pleura. While epithelial membrane antigen (EMA) and human milk fat globule-2 (HMFG-2) are each reported to stain the majority of cases of adenocarcinoma and mesothelioma, authors have reported that the pattern of staining for these two antibodies can be a useful discriminant. Both are described as revealing a membranous pattern of staining for mesothelioma and a cytoplasmic staining pattern in adenocarcinoma. Immunostains were performed on 23 cases of malignant mesothelioma and 22 cases of adenocarcinoma. For EMA, 78% of mesotheliomas and 86% of adenocarcinomas stained positively, and 65% of mesotheliomas vs. 14% of adenocarcinomas exhibited a membranous staining pattern. For HMFG-2, 9% of mesotheliomas and 50% of adenocarcinomas stained positively, and 4% of mesotheliomas vs. 9% of adenocarcinomas exhibited a membranous staining pattern. Membranous staining for HMFG-2 was not a useful criterion. In this series, a membranous pattern of staining for EMA had a sensitivity of 65% and a specificity of 86% for the identification of malignant mesothelioma.

Adenocarcinoma↗

PubMed1998

Mucosal iodine staining improves endoscopic visualization of squamous dysplasia and squamous cell carcinoma of the esophagus in Linxian, China.

BACKGROUND: In previous studies in the high risk population of Linxian, China, the majority of foci of high grade (moderate and severe) squamous dysplasia (HGD) and invasive squamous carcinoma (CA) of the esophagus were associated with endoscopically visible lesions that could be targeted for biopsy, but some foci of HGD were missed by routine endoscopic examination. This study examined whether spraying the mucosa with Lugol's iodine solution, which stains normal epithelium brown but leaves dysplasia and carcinoma unstained, could improve endoscopic detection and delineation of these lesions. METHODS: Two hundred twenty-five Linxian adults with balloon cytologic evidence of dysplasia or carcinoma underwent endoscopy. All visible lesions were described and photographed before and after staining with 1.2% Lugol's iodine solution. Biopsies were taken from all lesions visible before staining, from all unstained lesions (USLs) after applying the stain, and from representative control areas of stained mucosa. RESULTS: Two hundred fifty-three USLs and 255 control sites were biopsied. No complications occurred. Ninety-four biopsy sites contained HGD and 20 contained CA. Before staining, the sensitivity of visible lesions for identifying HGD or CA was 62%, and the specificity was 79%. After staining, the sensitivity of USLs for identifying HGD or CA was 96%, and the specificity was 63%. Eighty-eight percent of the HGD and CA lesions were larger or more clearly defined after staining. The diagnostic lesions in 17 of 31 patients with moderate dysplasia (55%), 8 of 35 patients with severe dysplasia (23%), and none of the 19 patients with invasive carcinoma (0%) were identified only after staining. CONCLUSIONS: Mucosal iodine staining improved endoscopic detection and delineation of HGD and CA in these patients. This simple technique is highly sensitive for identifying these precursor and invasive squamous lesions, and it should be used whenever optimal visualization of squamous mucosal abnormalities is required.

Adult↗

PubMed1998

Detection of coccoid Helicobacter pylori: light microscopical immunogold silver enhancing stain.

BACKGROUND: Helicobacter pylori (H. pylori) can be morphologically divided into spiral and coccoid forms. Although many different staining procedures for light microscopy have been developed to detect H. pylori, there are no reports that the coccoid forms can be specially identified by a staining method. The ability to identify the coccoid form of H. pylori by light microscopy would be important for studies evaluating its possible role in gastric disease. We examined whether both the coccoid and the spiral forms could be stained using anti-H. pylori antibody. METHODS: Specimens from the stomachs of dyspeptic patients with proven H. pylori infection and H. pylori from culture (1st and 4th day) were stained with the light microscopic immunogold silver enhancing stain using the rabbit IgG specific for H. pylori. Cultured H. pylori was also stained with electron microscopic immunogold staining method using the same antibody. The number of coccoid forms was counted by scanning electron microscopy. RESULTS: Biopsies and H. pylori from 6 patients were studied. H. pylori from both biopsy material and culture appeared as black organisms by light microscopic immunogold silver enhancing stain. The coccoid forms constituted 0.4% and 98.3% on days 1 and 4 of culture, respectively (by scanning electron microscopy). The antigen recognized by the anti-H. pylori antibody was located on the surface of the flagella, the cell wall, or in the cytoplasm by immunoelectron microscopy. CONCLUSION: This study shows that both spiral and coccoid forms of H. pylori can be detected by light microscopic immunogold silver enhancing stain using anti-H. pylori antibody. This simple stain is to be proven useful for studies aimed at understanding whether the coccoid form plays a role in disease.

Animals↗

PubMed1992

What is actually stained by rose bengal?

It has been believed that 1% rose bengal does not stain normal, healthy cells but rather stains degenerated or dead cells and mucous strands. In contrast to this conventional knowledge, we discovered that both commercial additive-containing and additive-free rose bengal solutions stained four different types of healthy cultured cells, including rabbit corneal epithelial cells. Rose bengal staining was rapid, dose dependent, predominantly nuclear, and detectable with the naked eye at concentrations as low as 0.05% and 0.025% for the commercial additive-containing or additive-free solutions, respectively, and with the fluorescence microscope at a concentration of 0.001%. It is surprising to discover that rose bengal is not a vital dye; after staining, cells actually lost vitality, as evidenced by instant morphologic changes, subsequent loss of cellular motility, cell detachment, and cell death. Such an intrinsic toxic effect was augmented by light exposure. The rose bengal staining of live as well as detergent-treated (Triton X-100) cells could be blocked by such tear components as mucin and albumin, suggesting that normally negative rose bengal staining is due to the protective function of the preocular tear film, ie, staining is not dictated by lack of cell vitality. These data indicate that rose bengal staining ensues whenever there is poor protection of surface epithelium by the preocular tear film; this represents a new interpretation for rose bengal stains seen in various ocular surface disorders.

Animals↗

PubMed1995

Initial lesion size as a predictive factor in determining the response of port-wine stains in children treated with the pulsed dye laser.

OBJECTIVE: To evaluate the response of facial port-wine stains in children to pulsed (450 microseconds) dye (577- or 585-nm) laser treatment based on the age of the patient and the size of the port-wine stain at the initiation of treatments. DESIGN: Case series. SETTING: Outpatient dermatology clinic at the University of Colorado School of Medicine, Denver. PATIENTS: A consecutive sample of 83 children who had facial port-wine stains that were treated in their entirety at each visit. INTERVENTION: Patients were treated with a pulsed (450 microseconds) dye (577- and 585-nm) laser. MAIN OUTCOME MEASURE: Decrease in size of the port-wine stain. RESULTS: Overall 18 (22%) of the 85 patients responded with complete clearing of their port-wine stains. The port-wine stains in 32% (7/22) of the patients who began treatment before 1 year of age responded with complete clearing of their port-wine stains compared with 18% (11/61) in older patients (P < .01). Fifteen (32%) of 47 patients with port-wine stains smaller than 20 cm2 at initial evaluation were totally cleared vs three (8%) of the 36 patients with port-wine stains larger than 20 cm2 (P < .05). This size effect is apparent at all ages. CONCLUSION: When considering treatment for facial port-wine stains with pulsed dye lasers, it is important to consider not only the age of the child at the beginning of treatments, but also the initial size of the lesion.

Adolescent↗

PubMed1988

Acetylcholinesterase fiber staining in the human hippocampus and parahippocampal gyrus.

The AChE fiber distribution within the human hippocampus and parahippocampal gyrus was studied in order to provide normative data for the examination of cholinergic fiberarchitecture in human pathology and to clarify the cytoarchitectonic organization of these structures. A modification of the Koelle method was used to stain temporal lobe serial sections from 6 neurologically normal human brains collected at autopsy. The hippocampal formation contains some of the densest staining of any cortical area. Regions with the heaviest concentrations of AChE fibers include a thin band along the inner edge of the molecular layer of the dentate gyrus (ml-DG) and parts of the CA2, CA3, and CA4 sectors of Ammon's horn. Staining is of intermediate intensity in the CA1 region. The subiculum (S) is more lightly stained than the CA fields. Staining in the parahippocampal gyrus is generally less dense than in the hippocampal formation. The most conspicuous feature of the human entorhinal cortex (EC) is the AChE-rich fiber patches seen overlapping the stellate cell islands in layer II. An additional band of relatively dense AChE staining is identified in layers IV-V. Prominent AChE-rich polymorphic neurons are present within the hilum of the dentate gyrus. The CA1/subiculum transition in Nissl preparation is characterized by an oblique interdigitation of CA1 cells. The transition from EC to prorhinal cortex occurs along the medial bank of the rhinal sulcus and is characterized by a band of AChE staining, which slopes obliquely away from layer II until it joins an intermediate pyramidal cell layer. Some comparisons with AChE staining in the monkey were made. The monkey has a similar pattern except in DG, where the intensely AChE staining band along the inner ml-DG is thicker and much more prominent. In comparison to the human, the monkey has more conspicuous AChE staining in the parasubicular region.

Acetylcholinesterase↗

PubMed1994

Immunocytochemical staining of c-erb B-2 oncogene in fine-needle aspirates of breast carcinoma: a comparison with tissue sections and other breast cancer prognostic factors.

The role of fine-needle aspiration (FNA) in breast cancer management is expanding to include prognostic in addition to diagnostic studies. This study was undertaken to compare the immunohistochemical staining of c-erbB-2, a breast cancer prognostic factor, on cytologic FNA smears of breast cancer with that on corresponding formalin-fixed, paraffin-embedded tissue sections of the same tumor and to correlate the c-erbB-2 expression with other known breast cancer prognostic factors. FNA smears (destained, alcohol-fixed, Pap-stained direct smears) and corresponding tissue sections (formalin-fixed, paraffin-embedded, unstained sections) from 36 primary breast carcinomas were stained immunohistochemically with c-erbB-2 antibody using an avidin-biotin procedure. Ten tumors (28%) showed strong positive staining for c-erbB-2 on the FNA smear and, of those ten, seven were positive on the corresponding tissue section and three were negative. In several of the cytology and tissue positive cases, staining on the tissue section was significantly weaker than on the FNA smear. Two cases involved treatment with preoperative chemotherapy; in one of those cases, tissue staining was weaker than cytologic staining, and, in the other case, both were negative. Correlation of c-erbB-2 staining with other prognostic factors showed an association between positive c-erbB-2 staining and high nuclear grade. Our results indicate that immunohistochemical staining for c-erbB-2 can be performed on destained FNA smears of breast carcinomas and may be more sensitive on the cytologic specimens than on formalin-fixed, paraffin-embedded tissue sections.

Biopsy, Needle↗

PubMed1977

FITC-labelled antibody staining of tropomyosin-containing fibrils in smooth, cardiac and skeletal muscle cells, prefusion myoblasts, fibroblasts, endothelial cells and 3T3 cells in culture.

FITC-labelled antibodies to purified chicken gizzard smooth muscle tropomyosin were prepared and used to stain muscle and non-muscle cells in culture. Skeletal muscle myoblasts stained both diffusely throughout the cytoplasm and in fine filamentous structures. Once myotubes developed the staining was localized exclusively in the I-band region of the myofibrils. Similarly, cardiac muscle cells stained in the I-band alone. Primary and subcultured smooth muscle cells, irrespective of their state of differentiation, stained exclusively in long, straight fibrils. The staining of the fibrils was interrupted with stained regions 1-2 micrometer long and unstained spacings 0.5 micrometer. Interrupted fibrils were also observed in fibroblasts and endothelial cells, however their staining reaction was very weak (almost indistinguishable from that with pre-immune serum) and they were few in number. 3T3 cells demonstrated moderate staining in interrupted fibrils. Sheaths of very fine fibrils staining with a similar intensity were also found throught the cytoplasm. Interruptions in these fine fibrils were often aligned to give the whole cell a striated appearance. Sheaths of fibrils were not found in the other cell types studied.

Animals↗

PubMed1979

Detection of HBsAg containing cells in liver biopsies by different stains and classification of positively reacting ground-glass hepatocytes.

The diagnostic significance of orcein, aldehydthionine, and chromotrope anilinblue stains for the demonstration of HBsAg containing hepatocytes was investigated in 602 unselected liver biopsies. Five types of specifically stained ground-glass hepatocytes (GGH) were distinguished: Type I showed a positive staining reaction of the cytoplasmic periphery (marginal GGH), type II a diffuse staining of the total cytoplasm (diffuse GGH). Type III contained round or oval globular positive cytoplasmic masses (globular GGH). Type IV showed only very small round, drop-like or sickle-shaped positive structures (spotty GGH). The GGH with fatty changes were designated as type V. In all carriers and patients with minimal hepatitis GGH, mostly type I and II, appeared in extensive clusters within the lobules. In chronic persistent hepatitis, there were moderately numerous, partly grouped, partly disseminated ground-glass hepatocytes of type II and III. In chronic active hepatitis there were only a few GGH of type IV. In acute viral hepatitis, there were no typical GGH, however, positively stained phagocytes were seen. The intracellular antigen localization and the intralobular distribution of GGH are considered to be the result of an immune reaction. Single so-called 'metabolic' GGH sometimes showed similar pictures. However, they could usually be distinguished from virus containing GGH because of their granular cytoplasmic structure and a lower staining intensity in the applied stains. Among the three stains the orcein stain yielded the best results. In some cases with HBsAg-positive chronic active hepatitis virus infection could not be proved by means of staining.

Biopsy↗