Transvaginal hydrolaparoscopy for diagnosis of tubal infertility

OBJECTIVE: Infertility problem affects more than 70 million couples worldwide, 5-15% of which are couples in their reproductive age. Less and less invasive endoscopic methods like transvaginal hydrolaparoscopy have been developed by technological progress. This method enables not only precise identification, but is now increasingly used for treatment of tubal and peritoneal factor pathology, which cause approximately 35 per cent of female infertility. AIM: Evaluation of transvaginal hydrolaparoscopy (HLTV) usefulness for diagnosis of tubal infertility comparing to standard laparoscopy and hysterosalpingography (HSG). Results: In evaluation of patent fallopian tubes results of HLTV and HSG examinations are coincide in 87%, while obstruction diagnosed in HSG is confirmed only in 37% during HLTV examination. Transvaginal hydrolaparoscopy and HSG have similar sensitivity and specificity in diagnosis of hydrosalpinx, which is up to 100% . In comparison with HLTV histerosalpingography is less effective in evaluation of peritubal dilatations and adhesions. Both laparoscopic surgery and transvaginal laparoscopy have the same high sensitivity in diagnostics of the fallopian tubes patency and hydrosalpinx, which is up to 100%. In evaluation of peritubal adhesions and dilatations the results are very similar. Conclusions: 1. HLTV is a highly useful method in evaluation of the fallopian tubes pathologies which is significantly more sensitive than HSG in evaluation of such lesions as peritubal adhesions and obstructed fallopian tubes. 2. HLTV is as effective as laparoscopy in evaluation of patency and lesions of the fallopian tubes. 3. HLTV is a less invasive method, much better tolerated than laparoscopy and more suitable for the group of overweight patients. 4. Final assessment of HTLV technique will be possible following performance of a greater number of studies, where the foregoing conclusions present only initial observations.     

Sequential Use of Wright's and Ziehl-Neelsen's Stains for Demonstrating Phagocytosis of Bacterial Spores

Nongerminating spores, germinating spores, and vegetative cells of Clostridium botulinum type A were observed during phagocytosis in the peritoneal fluid of white mice. Since phagocytes are easily ruptured by heat, the method described avoids heating, as this has been employed in conventional spore staining methods. A thin smear of the fluid is air dried on the slide for 2 hr, and stained by Wright's method: stain, 2 min; dilution water, 2 min; and rinsed; then in 0.005% methylene blue for 30 sec, and rinsed. This is followed by Ziehl-Neelsen's stain for 3-4 min and destained with 1: acetone-95% ethanol for 10 sec. The slide is rinsed, and Wright's staining repeated: stain 1 min, dilution 2-3 min; and thereafter washed about 5 ml of Wright's buffer. Blotting and air drying completes the staining. Non-germinating spores stain light red with a red spore wall, germinating spores are deep red throughout, vegetative cells are blue, and leucocytes show a dark purple nucleus and light blue cytoplasm.     

Chromoendoscopy with a Standard-Resolution Colonoscope for Evaluation of Rectal Aberrant Crypt Foci

Colorectal cancer (CRC) is the second most common cause of death worldwide. According to the theory by Vogelstein, colorectal carcinogenesis involves a series of successive changes in the normal colonic mucosa, starting with excessive proliferation and focal disorders of intestinal crypts, followed by adenoma and its subsequent malignant transformation. The first identifiable changes in CRC carcinogenesis are aberrant crypt foci (ACF). ACF are invisible during routine colonoscopy yet are well identifiable in chromoendoscopy using methylene blue or indigo carmine. High-resolution colonoscopes are used for assessment of ACF. The aim of the present study was to evaluate the usefulness of standard-resolution colonoscopy for identification of rectal ACF. The following parameters were evaluated: duration of chromoendoscopy of a given rectal segment, type of ACF, sensitivity and specificity of endoscopy combined with histopathological evaluation. The mean duration of colonoscopy and chromoendoscopy was 26.8 min. In the study population, typical ACF were found in 73 patients (p = 0.489), hyperplastic ACF in 49 (p = 0.328), and dysplastic ACF in 16 patients (p = 0.107). Mixed ACF were observed in 11 individuals (p = 0.073). The sensitivity of the method was found to be 0.96 whereas its specificity 0.99.Identification of rectal ACF using standard-resolution colonoscopy combined with rectal mucosa staining with 0.25% methylene blue is characterised by high sensitivity and specificity.     

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.     

Methods for the Standardization of Biological Stains Part V

In this paper are given the methods for determining the suitability of certain dyes of the pyronin, thiazin, oxazin, azin and natural dye groups for certification by the Commission on Standardization of Biological Stains. These methods have been developed by the Commission in cooperation with the Color and Farm Waste Division, Bureau of Chemistry and Soils, U. S. Department of Agriculture. The dyes for which the methods are given in the present paper are: Pyronin G, pyronin B, neutral red, safranin, nigrosin water-soluble, brilliant cresyl blue, cresyl violet, Nile blue A, thionin, methylene blue, methylene azure (azure A), azure C, toluidine blue O, indigo carmin (indigotine) and carmin. For each of these dyes methods are discussed under the following headings: (1) identification or qualitative examination; (2) quantitative analysis; and (3) biological tests.     

Methods for the Standardization of Biological Stains Part V

In this paper are given the methods for determining the suitability of certain dyes of the pyronin, thiazin, oxazin, azin and natural dye groups for certification by the Commission on Standardization of Biological Stains. These methods have been developed by the Commission in cooperation with the Color and Farm Waste Division, Bureau of Chemistry and Soils, U. S. Department of Agriculture. The dyes for which the methods are given in the present paper are: Pyronin G, pyronin B, neutral red, safranin, nigrosin water-soluble, brilliant cresyl blue, cresyl violet, Nile blue A, thionin, methylene blue, methylene azure (azure A), azure C, toluidine blue O, indigo carmin (indigotine) and carmin. For each of these dyes methods are discussed under the following headings: (1) identification or qualitative examination; (2) quantitative analysis; and (3) biological tests.     

The influence of NO synthase inhibitor and free oxygen radicals scavenger--methylene blue--on streptozotocin-induced diabetes in rats

The excessive production of nitric oxide (NO) and the subsequent increase of local oxidative stress is suggested as one of the pathophysiological mechanisms of streptozotocin-induced diabetes. It was reported that the administration of NO synthase inhibitors partially attenuated the development of streptozotocin-induced diabetes and reduced hyperglycaemia. Here we have studied the influence of methylene blue, which combines the properties of NO synthase inhibitor with antioxidant effects. The experiments were performed on male rats divided into four groups: control, diabetic (single dose of 70 mg of streptozotocin/kg i.p.), methylene blue (50 mg/kg in the food) and diabetic simultaneously fed with methylene blue. After 45 days the experiments were discontinued by decapitation. Serum glycaemia, glycated haemoglobin and oxidative stress parameters (plasma malondialdehyde concentration and erythrocyte superoxide dismutase activity) were significantly higher in the diabetic group. Simultaneous methylene blue administration partially reduced glycaemia and glycated haemoglobin, but did not decrease oxidative stress. We conclude that NO synthase inhibitor methylene blue partially attenuates the development of streptozotocin-induced diabetes in male rats, but does not reduce the development of oxidative stress in the diabetic group.     

In vivo and in vitro decolorization of synthetic dyes by laccase from solid state fermentation with Trametes sp. SYBC-L4

Synthetic decolorization of dyes through solid cassava residue substrate fermentation with Trametes sp. SYBC-L4 via in vivo and in vitro processes was investigated in this study. Effects of pH and mediator (1-hydroxybenzotriazole, HBT) concentration on dyes decolorization were evaluated. In vitro, decolorization ratios of dyes differed considerably in pH and increased with the increasing of HBT concentration. Crude laccase (50 U/L) derived from Trametes sp. SYBC-L4 decolorized 67.91 ± 1.25 % Congo red (100 mg/L), 94.58 ± 1.05 % aniline blue (100 mg/L) and 99.02 ± 0.54 % indigo carmine (100 mg/L) with 2.5 mM HBT at pH 4.5 in 36 h of incubation. In vivo, decolorization ratios of dyes were not enhanced by usage of the mediator. After 10 days of fermentation, decolorization ratio of Congo red (1,000 mg/kg), aniline blue (1,000 mg/kg) and indigo carmine (1,000 mg/kg) was 57.82 ± 0.84, 92.53 ± 1.12 and 97.26 ± 1.92 % without the usage of mediator at pH 4.5, respectively. Moreover, there was no obvious difference between the in vivo decolorization of aniline blue and indigo carmine in the pH range of 3.0–9.0. Results showed that Trametes sp. SYBC-L4 had great potential to be used for dyes decolorization via in vivo and in vitro processes. Moreover, in terms of pH range and mediator, in vivo decolorization with Trametes sp. SYBC-L4 was more advantageous since laccase mediator was needless and the applicable range of pH was broader.     

Uptake of erythrocyte-associated component of blood testosterone and corticosterone to rat brain

To study transport of steroids by erythrocytes, the tissue uptake of erythrocyte-associated testosterone and corticosterone was studied in vivo using a single injection technique into the carotid artery of rats. A brain uptake index (BUI) was calculated by dividing the ratio of [3H]steroid to [14C]butanol (internal reference) in the brain tissue by that in the injection material, and multiplying by 100%. BUIs of testosterone and corticosterone in an erythrocyte suspension were 131 +/- 3% (mean +/- SE, n = 6) and 57.0 +/- 2.7% (n = 6), respectively, which were greater than those in buffer (100 +/- 4%; n = 4, P less than 0.01 and 39.8 +/- 4.6%; n = 4, P less than 0.01, respectively). The erythrocyte accounted for 83.9% and 76.7% of the total testosterone and corticosterone delivered to the tissues, respectively, when calculated on the assumption that the BUIs of steroid in buffer and in the supernatant of an erythrocyte suspension are the same. BUIs of corticosterone in hemolysate and in a suspension of erythrocyte plasma membranes (60.8 +/- 7.0%; n = 4 and 69.5 +/- 3.7%; n = 4, respectively) were also greater than those in buffer (P less than 0.05 and P less than 0.01, respectively). Our results suggest that the erythrocyte-associated component of testosterone and corticosterone are delivered to the tissue of rat brain, and that their membranes may play a major role in their capacity to transport steroids to the tissues.     

PGE2 and PGF2α release by human peritoneal macrophages in endometriosis

Objective: To test for differences in the amount and activity of peritoneal macrophages present in the peritoneal fluid of women with, and without endometriosis using prostaglandin release by macrophages in culture as a marker.Patients: Women of reproductive age undergoing laparoscopy for infertility or chronic pelvic pain with postoperative diagnosis of endometriosis and women undergoing laparoscopy for sterilization.Methods: Peritoneal fluid was aspirated during laparoscopy, volume was recorded, macrophages were isolated via a Ficoll Paque gradient and kept in primary culture. PGE₂ and PGF_2α release of the cells were measured before and after stimulation with zymosan.Results: Women with endometriosis had significantly more peritoneal macrophages than controls. Peritoneal macrophages of women with endometriosis released significantly more PGE₂ than those of the control group: 8.4 ± 2.0 versus 1.4 ± 0.4 ng/ml/10⁶cells (mean ± SEM, p=0.0005) and PGF_2α : 10 ± 4.3 (endometriosis) versus 1.8 ± 0.4 (control) ng/ml/10⁶cells (mean ± SEM, p = 0.045).Conclusion: There is a significant increase in the amount of prostaglandins released by peritoneal macrophages from women with endometriosis. These prostaglandins might alter uterine and tubal contractility, thereby affecting fertility.     

Azoreductase from alkaliphilic Bacillus sp. AO1 catalyzes indigo reduction

Indigo is an insoluble blue dye historically used for dyeing textiles. A traditional approach for indigo dyeing involves microbial reduction of polygonum indigo to solubilize it under alkaline conditions; however, the mechanism by which microorganisms reduce indigo remains poorly understood. Here, we aimed to identify an enzyme that catalyzes indigo reduction; for this purpose, from alkaline liquor that performed microbial reduction of polygonum indigo, we isolated indigo carmine-reducing microorganisms. All isolates were facultative anaerobic and alkali-tolerant Bacillus spp. An isolate termed AO1 was found to be an alkaliphile that preferentially grows at pH 9.0–11.0 and at 30–35 °C. We focused on flavin-dependent azoreductase as a possible enzyme for indigo carmine reduction and identified its gene (azoA) in Bacillus sp. AO1 using homology-based strategies. azoA was monocistronic but clustered with ABC transporter genes. Primary sequence identities were < 50% between the azoA product (AzoA) and previously characterized flavin-dependent azoreductases. AzoA was heterologously produced as a flavoprotein tolerant to alkaline and organic solvents. The enzyme efficiently reduced indigo carmine in an NADH-dependent manner and showed strict specificity for electron acceptors. Notably, AzoA oxidized NADH in the presence, but not the absence, of indigo. The reaction rate was enhanced by adding organic solvents to solubilize indigo. Absorption spectrum analysis showed that indigo absorption decreased during the reaction. These observations suggest that AzoA can reduce indigo in vitro and potentially in Bacillus sp. AO1. This is the first study that identified an indigo reductase, providing a new insight into a traditional approach for indigo dyeing.

     

Experimental adhesion model: effect of viscosities of fluids put in the peritoneal cavity on preventing peritoneal adhesions.

In this study we assessed the effectiveness of fluid viscosities placed in the peritoneal cavity to prevent postoperative peritoneal adhesions. Thirty-six Wistar albino female rats (average weight: 160 +/- 30 g, average age: 6.5 months) were divided into three groups of equal number. A standard adhesion pattern was formed in each group. Then, 3 ml isotonic sodium chloride solution (relative viscosity value: 1) was added into the peritoneal cavity of group 1; 3 ml standard 6% hydroxy ethyl starch solution (HES) (relative viscosity value: 2.9) was added into the peritoneal cavity of group 2; and a standard HES solution that was concentrated by dehydration (relative viscosity value: 249.7) was added into the peritoneal cavity of group 3. All rats were sacrificed on postoperative day 10 and the adhesions that formed were graded. In group 1, grade-3 adhesions developed in 9 (75%) rats, and grade-2 developed in 3 (25%) rats. In group 2, grade-3 adhesions developed in 1 (8.3%) rat, grade-2 developed in 6 (50%) rats, and grade-1 developed in 5 (41.6%) rats; in group 3, grade-3 adhesions developed in 9 (75%) rats, and grade-2 developed in 3 (25%) rats. The adhesion scores of group 3 and group 1 were equal to each other (P=1), while the adhesion score of group 2 was significantly less (chi(2): 18.23, P<0.001). Increasing the viscosity of fluids that are inserted in the peritoneal cavity may reduce the formation of postoperative peritoneal adhesions till a critical value of unknown viscosity is achieved. The mechanism behind this process remains unclear.     

Differential Cytophysiological Diagnosis of Cancerous and Normal Tissues

A method is offered for he differential diagnosis of cancer cells. It depends on the use of methylene blue decolorized with sodium thiosulfate (denoted here HLM, i.e. “hyposulfite methylene blue”); this is prepared by dissolving 800 mg. sodium thiosulfate in 10 ml. of 0.1% aqueous methylene blue and adding 3-5 drops of dilute (1:3) HCl. Frozen sections are treated with this reagent for 2-3 minutes, rinsed with a large amount of distilled water, then stained 2-3 minutes with 0.05% aqueous acid fuchsin. Staining should be performed in a darkened room. If all due precautions are observed, normal tissue appears blue, malignant tissue red.     

Effects of zinc deficiency and supplementation on some hematologic parameters of rats performing acute swimming exercise

The aim of the study was to investigate how zinc deficiency and supplementation effect some hematologic parameters of rats performing swimming exercise. Forty adult male Spraque-Dawley rats were divided into 4 groups, zinc deficient swimming group (Group 1, n=10, zinc supplemented swimming group (Group 2, n=10), swimming control group (Group 3, n=10), and control group (Group 4, n=10). Blood samples were taken by decapitation and analyzed for the determination of erythrocyte, hemoglobin level, hematocrit, leukocyte, lymphocyte, platelet count and plasma zinc level at the end of the 4 week experiment. Erythrocyte count of group 1 was the lowest whereas erythrocyte count in group 3 was significantly lower than that in group 2 and 4 (p<0.05). Hemoglobin level of group 1 was significantly lower than that of groups 2 and 4 (p<0.05). Hematocrit was significantly lower in both group 1 and group 3 than both groups 2 and 4 (p<0.05). Lymphocyte count in group 2 was significantly higher than in all other groups (p<0.05). Platelet counts in group 2 was significantly lower than in all other groups (p<0.05). Our findings suggest that zinc deficiency effects the hematologic parameters mentioned negatively whereas zinc supplementation has a positive influence.     

贮存式自体成分输血对胃肠肿瘤根治术患者T淋巴细胞亚群、血液流变学和预后的影响

摘要 目的：研究贮存式自体成分输血对胃肠肿瘤根治术患者T淋巴细胞亚群、血液流变学以及预后的影响。方法：选取2016年12月~2017年12月我院收治的95例行胃肠肿瘤根治术的胃肠肿瘤早期患者作为研究对象。按随机数表法分为A组（n=47,贮存式自体成分输血）和B组(n=48,异体成分输血)。比较两组患者血常规指标[红细胞计数(RBC)、血红蛋白（Hb）、血细胞比容（Hct）]、免疫功能指标（CD3⁺、CD4⁺、CD8⁺、CD4⁺/CD8⁺）、血液流变学指标[红细胞沉降率、平均血液黏度、红细胞刚性指数及红细胞变形指数]的变化。随访患者2年,采用Kaplan-Meier曲线分析两组预后情况。结果：术前至术后7 d 时间段A组的CD3⁺、CD4⁺水平及CD4⁺/CD8⁺呈先降低后升高趋势,且术后7 d已恢复至术前水平;B组术后1 d、3 d、7 d的CD3⁺、CD4⁺水平及CD4⁺/CD8⁺明显低于A组(P＜0.05),两组CD8⁺水平相比无差异(P＞0.05) 。两组术后7 d的RBC、Hct、Hb分别较输血前下降（P＜0.05）,但两组间比较差异无统计学意义(P＞0.05);两组患者术后7 d的红细胞沉降率均升高,但A组低于B组(P＜0.05) ,两组患者术后的红细胞变形指数、平均血液黏度和红细胞刚性指数比较无差异 (P＞0.05) 。Kaplan-Meier检验结果显示,A组较B组生存率明显升高（P＜0.05）。结论：贮存式自体成分输血对胃肠肿瘤根治术患者红细胞沉降率、细胞免疫功能的影响程度较异体成分输血减轻,且贮存式自体成分输血可以明显提高患者的术后生存率。     

Effect of fluconazole on phagocytic response of polymorphonuclear leukocytes in a rat model of acute sepsis

Recently, fluconazole (FLZ) has been shown to improve survival and reduce multiorgan failure in experimental and clinical septic shock. The mechanism by which FLZ affords protection against sepsis remains obscure. This study examines the effect of FLZ on phagocytic activity of polymorphonuclear leukocytes (PMNs) in a rat model of septic shock by inducing fecal peritonitis in male Wistar rats using intraperitoneal instillation (1 mL/kg) of fecal suspension in saline (1:1 w/v). Sham control rats received sterile fecal suspension and vehicle treatment. FLZ was administered in the doses of 0, 3, 10, and 30 mg/kg by gavage 30 minutes before fecal instillation. The samples of peritoneal fluid were collected 8 hours following fecal inoculation for the evaluation of phagocytic response of PMNs using zymosan-induced luminol-dependent chemiluminescence (CL). Fecal peritonitis caused massive infiltration of PMNs in the peritoneal cavity (ANOVA F(4,45) = 6.322, P < .001). Although FLZ reduced the infiltration of PMNs, this effect was neither significant nor dose dependent. The actual CL response was significantly higher in the peritoneal fluid of rats subjected to peritonitis, which was significantly and dose dependently attenuated by FLZ treatment (ANOVA F(4,45) = 11.048, P < .001). Normalization of CL response for 1000 PMNs revealed that FLZ dose-dependently albeit insignificantly reduced the activity of PMNs. The high dose of FLZ caused 2.29-fold decrement in the area under curve (AUC) pertaining to cumulative CL response. The findings of this study suggest that FLZ protects rats against septic shock by inhibiting PMN-mediated inflammatory cascade without compromising their phagocytic activity.     

Functional aspects of peritoneal macrophages in endometriosis of women

Peritoneal fluid was collected in the periovulatory phase of the cycle from 25 women undergoing laparoscopy. Endometriosis was diagnosed in 13 patients (AFS score 1, N = 9; AFS score 2, N = 4) and 12 patients without endometriosis served as controls. In endometriosis patients the total peritoneal fluid cell number and cell concentration was significantly higher than in controls, indicating peritoneal irritation by endometrial implants. Peritoneal fluid macrophages in patients with endometriosis showed significantly increased erythrophagocytosis and lower chemiluminescence than in controls, suggesting an advanced differentiation of the macrophages in endometriosis patients. The macrophages in this stage of differentiation may interfere with gametes and embryos and thus contribute to endometriosis-associated subfertility.     

全氟显4D-HyCoSy与3D-HyCoSy对输卵管性不孕症患者的诊断效果评价

摘要 目的：探讨与评价全氟显子宫输卵管四维超声造影（4D-HyCoSy）与经阴道三维超声输卵管造影（3D-HyCoSy）对输卵管性不孕症患者的诊断效果。方法：选择2018年8月到2021年11月选择在本院诊治的女性不孕症患者84例作为研究对象,都给予全氟显4D-HyCoSy与3D-HyCoSy诊断,记录图像质量与不良反应发生情况。金标准为腹腔镜检查,判断诊断效果。结果：在84例患者中,全氟显4D-HyCoSy的图像质量优良率为100.0 %,高于3D-HyCoSy的92.9 %（P<0.05）。全氟显4D-HyCoSy诊断为输卵管通畅14例,输卵管通而不畅23例,输卵管阻塞47例。3D-HyCoSy诊断：22例输卵管通畅,21例输卵管通而不畅,41例输卵管阻塞。腹腔镜诊断：15例输卵管通畅,21例输卵管通而不畅,48例输卵管阻塞。全氟显4D-HyCoSy、3D-HyCoSy对输卵管性不孕症患者的诊断敏感性为100.0 %（36/36）、97.2 %（35/36）,特异性为97.9 %（47/48）、83.3 %（40/48）。84例患者在全氟显4D-HyCoSy、3D-HyCoSy检查期间发生的不良反应主要为阴道出血、恶心呕吐、腹部疼痛等,不良反应发生率对比无差异（P>0.05）。全氟显4D-HyCoSy对输卵管性不孕症患者的诊断敏感性与3D-HyCoSy对比无差异（P>0.05）,诊断特异性高于3D-HyCoSy（P<0.05）。结论：相对于3D-HyCoSy,全氟显4D-HyCoSy在输卵管性不孕症患者的应用能提高图像质量优良率,具有很好的安全性,还具有更好的诊断特异性。     

A Basic Fuchsin and Alkalinized Methylene Blue Rapid Stain for Epoxyembedded Tissue

Sections 1 μ thick of epoxy-embedded, OsO₄-fixed tissues were stained with 4% aqueous basic fuchsin at 70 C for 1 min, rinsed well and destained, also at 70 C, for 1 min. A 2% aqueous methylene blue solution, alkalinized to pH 12.5 by mixing 1 N NaOH with the dye on the slide in the proportion of about 2:1, was then allowed to act for 2 min at 23-27 C. The stain was rinsed off the slide, and the preparation air dried before applying a mounting medium and cover glass. The mounting medium consisted of immersion oil sealed with epoxy household cement. Stains had not faded after 1 yr. The method is simple, rapid (total time 4-5 min), and provides sharp contrast between cellular and connective tissue components.