Activation of the IGF1 receptor stimulates glycogen synthesis by mink uterine epithelial cells.

Glycogen synthesis by mink uterine epithelial cells is stimulated by estradiol (E2 ) during estrus, although the mechanism/s through which the steroid promotes glycogen accumulation are unknown. Our aim was to determine if insulin is required for E2 induced glycogen synthesis by an immortalized mink uterine epithelial cell line (GMMe). We show that the cells expressed the genes for glycogen metabolizing enzymes (hexokinase 1, glucose-6-phosphatase 3, glycogen synthase 1, and glycogen phosphorylase-muscle), receptors for insulin, insulin-like growth factor 1 and E2 (Esr1). Interestingly, treatment of cells with E2 alone failed to stimulate glycogen production, whereas supraphysiological concentrations of insulin (50 µg/ml) only, significantly increased glycogen content. Moreover, insulin + E2 increased glycogen content when compared to insulin alone (p < 0.05), an affect that was blocked when cells were treated with the pure E2 receptor antagonist ICI 182,780. Glycogen synthesis in response to insulin was significantly inhibited when cells were pre-treated with picropodophyllotoxin, an IGF1R antagonist. Treatment of cells with LY294002, a phosphatidylinositol 3-kinase (PI3K) antagonist, blocked insulin's effects on glycogen production whereas treatment with U0126, an inhibitor of mitogen activated kinase-kinase (MEK1/2) was without effect. These findings suggest to us that the affects of E2 on glycogen synthesis by GMMe cells is mediated through Esr1 and increased responsiveness of the cells to insulin. Because picropodophylotoxin blocked the effects of insulin on glycogen production, and both insulin and IGF1 act through PI3K, it is possible that IGF1 plays a role in glycogen production by these cells.

Generalized Safety and Efficacy of Simplified Intravenous Thrombolysis Treatment (SMART) Criteria in Acute Ischemic Stroke: The MULTI SMART Study.

BACKGROUND: Common intravenous recombinant tissue plasminogen activator (IV rt-PA) exclusion criteria may substantially limit the use of thrombolysis. Preliminary data have shown that the SMART (Simplified Management of Acute stroke using Revised Treatment) criteria greatly expand patient eligibility by reducing thrombolysis exclusions, but they have not been assessed on a large scale. We evaluated the safety and efficacy of general adoption of SMART thrombolysis criteria to a large regional stroke network. METHODS: Retrospective analysis of consecutive patients who received IV thrombolysis within a regional stroke network was performed. Patients were divided into those receiving thrombolysis locally versus at an outside hospital. The primary outcome was modified Rankin Scale score (≤1) at discharge and the main safety outcome was symptomatic intracranial hemorrhage (sICH) rate. RESULTS: There were 539 consecutive patients, and 50.5% received thrombolysis at an outside facility. Ninety percent of the patients possessed common conventional IV rt-PA contraindications. There were no significant differences between local and network treated patients in favorable outcome (45.4% versus 37.4%; odds ratio [OR], .72; P > .09), mortality (9% versus 14%; OR, 1.6; P > .07), or sICH rate (2.6% versus 5.1%; OR, 2.0; P = .13). Multivariate analysis showed no association between receiving IV rt-PA at an outlying spoke hospital and higher rate of sICH or worse outcome at discharge. CONCLUSIONS: Generalized application of SMART criteria is safe and effective. Widespread application of these criteria could substantially increase the proportion of patients who might qualify for treatment.

Uterine glycogen metabolism in mink during estrus, embryonic diapause and pregnancy.

We have determined uterine glycogen content, metabolizing enzyme expression and activity in the mink, a species that exhibits obligatory embryonic diapause, resulting in delayed implantation. Gross uterine glycogen concentrations were highest in estrus, decreased 50% by diapause and 90% in pregnancy (P ≤ 0.05). Endometrial glycogen deposits, which localized primarily to glandular and luminal epithelia, decreased 99% between estrus and diapause (P ≤ 0.05) and were nearly undetectable in pregnancy. Glycogen synthase and phosphorylase proteins were most abundant in the glandular epithelia. Glycogen phosphorylase activity (total) in uterine homogenates was higher during estrus and diapause, than pregnancy. While glycogen phosphorylase protein was detected during estrus and diapause, glycogen synthase was almost undetectable after estrus, which probably contributed to a higher glycogenolysis/glycogenesis ratio during diapause. Uterine glucose-6-phosphatase 3 gene expression was greater during diapause, when compared to estrus (P ≤ 0.05) and supports the hypothesis that glucose-6-phosphate resulting from phosphorylase activity was dephosphorylated in preparation for export into the uterine lumen. The relatively high amount of hexokinase-1 protein detected in the luminal epithelia during estrus and diapause may have contributed to glucose trapping after endometrial glycogen reserves were depleted. Collectively, our findings suggest to us that endometrial glycogen reserves may be an important source of energy, supporting uterine and conceptus metabolism up to the diapausing blastocyst stage. As a result, the size of uterine glycogen reserves accumulated prior to mating may in part, determine the number of embryos that survive to the blastocyst stage, and ultimately litter size.

Reduced clopidogrel metabolism in a multiethnic population: prevalence and rates of recurrent cerebrovascular events.

BACKGROUND: Concern has recently been raised over the possibility of a reduced efficacy of clopidogrel because of genetic variations in cytochrome P450, family 2, subfamily C, polypeptide 19 (CYP2C19) metabolism. A black box warning from the US Food and Drug Administration recommends that all patients be tested. It has been estimated that approximately 3% (range 2-14%) of the population are poor metabolizers, but few data are available for cerebrovascular patients. The objective of this study is to evaluate the frequency and effects of variability in CYP2C19 metabolism in patients with cerebrovascular disease. METHODS: A retrospective review of all patients with stroke and transient ischemic attack (TIA) tested for the clopidogrel CYP2C19 genotype was performed, with a collection of data including race/ethnicity, CYP2C19 status, and the presence of recurrent vascular events. RESULTS: A total of 53 cerebrovascular patients were tested, consisting of 5.7% poor (n = 3), 26.4% intermediate (n = 14), 62.3% extensive (n = 33), 3.8% indeterminate (n = 2), and 1.9% "mixed ultra rapid and poor" (n = 1) metabolizers. Only 10 of 38 white patients (26.3%; 95% confidence interval [CI] 0.14-0.42) were intermediate or poor metabolizers, compared with 7 of 15 (46.7%; 95% CI 0.25-0.70) nonwhites. Of 43 patients treated with clopidogrel, 3 of 27 extensive metabolizers (11.1%; 95% CI 0.04-0.28) had recurrent cerebrovascular events compared with 33.3% of intermediate metabolizers (4/12; 95% CI 0.14-0.61) and 50% of poor metabolizers (1/2; 95% CI 0.09-0.90). CONCLUSIONS: These data suggest that the proportion of poor/intermediate clopidogrel metabolizers in cerebrovascular patients is comparable to cardiovascular studies and these patients may have an increased risk of recurrent cerebrovascular events. Routine CYP2C19 testing may be warranted.

Glycogen metabolism in mink uterine epithelial cells and its regulation by estradiol, progesterone and insulin.

Glycogen content in mink uterine glandular and luminal epithelia (GE and LE) is maximal during estrus and is depleted before implantation while embryos are in diapause. Uterine glycogen synthesis in vivo is stimulated by estradiol (E2) while its mobilization is induced by progesterone (P4). Nevertheless, treatment of an immortalized mink uterine epithelial cell line (GMMe) with E2 did not affect glycogen production. Interestingly, insulin alone significantly increased synthesis of the nutrient and glycogen content in response to insulin + E2 was greater than for insulin alone. Our objectives were to determine: 1) If insulin receptor protein (INSR) is expressed by mink uterine GE and LE in vivo and if the amount differs between estrus, diapause and pregnancy; 2) if E2, P4 or insulin regulate insulin receptor gene (Insr) expression by GMMe cells, and 3) if E2 and P4 act independently to regulate glycogen metabolism by GMMe cells and/or if their effects are mediated in part through the actions of insulin. The mean (±S.E.) percent INSR content of uterine epithelia was greatest during diapause (GE: 15.65 ± 0.06, LE:16.56 ± 1.25), much less during pregnancy (GE: 2.53 ± 0.60, LE:2.25 ± 0.32) and barely detectable in estrus (GE: 0.03 ± 0.01, LE:0.02 ± 0.01). Glycogen concentrations in GMMe cells increased 10-fold in response to insulin and 20-fold with insulin + E2 when compared to controls. Expression of Insr was increased 2-fold by insulin and insulin + E2 when compared to controls and there was no difference between the two hormone treatments, indicating that E2 does not increase Insr expression in insulin-treated cells. To simulate E2-priming, cells were treated with Insulin + E2 for 24 h, followed by the same hormones + P4 for the second 24 h (Insulin + E2 → P4) which resulted in Insr and glycogen levels not different from controls. Similarly, cells treated with Insulin + P4 resulted in glycogen concentrations not different from controls. We conclude that the glycogenic actions of E2 on GMMe cells are due to increased responsiveness of the cells to insulin, but not as a result of up-regulation of the insulin receptor. Glycogen mobilization in response to P4 was the result of decreased glycogenesis and increased glycogenolysis occurring concomitantly with reduced Insr expression. Mink uterine glycogen metabolism appears to be regulated in a reproductive cycle-dependent manner in part as a result of the actions of E2 and P4 on cellular responsiveness to insulin.

Estradiol stimulates glycogen synthesis whereas progesterone promotes glycogen catabolism in the uterus of the American mink (Neovison vison).

Glycogen synthesis by mink uterine glandular and luminal epithelia (GE and LE) is stimulated by estradiol (E2 ) during estrus. Subsequently, the glycogen deposits are mobilized to near completion to meet the energy requirements of pre-embryonic development and implantation by as yet undetermined mechanisms. We hypothesized that progesterone (P4 ) was responsible for catabolism of uterine glycogen reserves as one of its actions to ensure reproductive success. Mink were treated with E2 , P4 or vehicle (controls) for 3 days and uteri collected 24 h (E2 , P4 and vehicle) and 96 h (E2 ) later. To evaluate E2 priming, mink were treated with E2 for 3 days, then P4 for an additional 3 days (E2 →P4 ) and uteri collected 24 h later. Percent glycogen content of uterine epithelia was greater at E2 + 96 h (GE = 5.71 ± 0.55; LE = 11.54 ± 2.32) than E2 +24 h (GE = 3.63 ± 0.71; LE = 2.82 ± 1.03), and both were higher than controls (GE = 0.27 ± 0.15; LE = 0.54 ± 0.30; P < 0.05). Treatment as E2 →P4 reduced glycogen content (GE = 0.61 ± 0.16; LE = 0.51 ± 0.13), to levels not different from controls, while concomitantly increasing catabolic enzyme (glycogen phosphorylase m and glucose-6-phosphatase) gene expression and amount of phospho-glycogen synthase protein (inactive) in uterine homogenates. Interestingly, E2 →P4 increased glycogen synthase 1 messenger RNA (mRNA) and hexokinase 1mRNA and protein. Our findings suggest to us that while E2 promotes glycogen accumulation by the mink uterus during estrus and pregnancy, it is P4 that induces uterine glycogen catabolism, releasing the glucose that is essential to support pre-embryonic survival and implantation.

Microneurosurgical Clip Ligation of Acutely Ruptured Cerebral Aneurysm Immediately Preceded by Intentional Subtotal Endovascular Coil Embolization Under a Single Anesthesia: Observations Using a Deliberate Combined Sequential Treatment Strategy in 13 Cases.

BACKGROUND: Endovascular coil embolization and craniotomy with clip ligation are the 2 most commonly used treatments for ruptured cerebral aneurysm. Although coiling maintains the advantages of brevity and complete avoidance of brain retraction and manipulation, clipping offers the benefits of decompression of the injured brain and lower rates of aneurysm recurrence. A combined, immediately sequential treatment strategy for acutely ruptured cerebral aneurysm that simultaneously maximizes the advantages of both techniques, while minimizing their respective disadvantages, may be a useful paradigm. OBJECTIVE: To demonstrate the complementarity of clipping and coiling in acutely ruptured cerebral aneurysm. METHODS: Patients with ruptured anterior circulation cerebral aneurysm standing to benefit from brain decompression were treated by a combination of coiling and microneurosurgery in rapid succession, under the same general anesthetic. Surgery consisted of clipping of the aneurysm via either craniotomy or craniectomy with expansion duraplasty in all cases, and ventriculostomy in selected cases. RESULTS: Coil embolization of the ruptured aneurysm was carried out rapidly and improved the efficiency of subsequent clipping by allowing early unequivocal identification of the aneurysm dome and decreased brain retraction, reducing risk of intraoperative rupture and obviating temporary occlusion. All aneurysms were shown eliminated by postoperative cerebral angiography. CONCLUSIONS: A deliberate combined treatment strategy that uses clipping immediately preceded by subtotal coiling under a single anesthetic may be ideal for selected ruptured cerebral aneurysms, takes advantage of the unique strengths of both techniques, makes both techniques easier, and maximizes opportunity for brain protection against delayed complications in the prolonged aftermath of aneurysmal subarachnoid hemorrhage.

A diffusion approach to labeling rows and columns in an irregular array.

A robust algorithm is presented for labeling rows and columns in an irregular array. The algorithm is based on hierarchical pattern matching to a local lattice, which is used as a template. Starting from the best local match, the pattern is expanded hierarchically to encompass the entire array. An application to labeling digitized images of an array of tissue sections mounted on a microscope slide is discussed.

Glycine increases preimplantation development of mouse oocytes following vitrification at the germinal vesicle stage.

Ice-free cryopreservation, referred to as vitrification, is receiving increased attention in the human and animal assisted reproduction. However, it introduces the detrimental osmotic stress by adding and removing high contents of cryoprotectants. In this study, we evaluated the effects of normalizing cell volume regulation by adding glycine, an organic osmolyte, during vitrification of mouse germinal vesicle stage oocyte and/or subsequent maturation on its development. The data showed that glycine supplementation in either vitrification/thawing or maturation medium significantly improved the cytoplasmic maturation of MII oocytes manifested by spindle assembly, chromosomal alignment, mitochondrial distribution, euploidy rate, and blastocyst development following fertilization in vitro, compared to the control without glycine treatment. Furthermore, glycine addition during both vitrification/thawing and maturation further enhanced the oocyte quality demonstrated by various markers, including ATP contents and embryo development. Lastly, the effect of anti-apoptosis was also observed when glycine was added during vitrification. Our result suggests that reducing osmotic stress induced by vitrification could improve the development of vitrified mouse oocyte.

Fecal progestin concentrations as an indicator of reproductive success in American Mink.

Efforts to increase mink reproductive success (live births and litter sizes) can be partly assessed by measurement of blood progesterone levels. However, the stress of blood sampling increases the incidence of failed matings, aborted fetuses and death of the dam. We have therefore non-invasively measured fecal progesterone metabolite (progestin) concentrations during the reproductive cycle of mink. We tested the hypothesis that fecal progestin concentrations during the window of implantation (late March-early April) will, (1): be higher for whelping than non-whelping mink, and (2): be higher for mink mated multiple times, compared to single matings. Mink were mated once (March 3), twice (March 3 and 10) or three times (March 3, 10 and 11) and fecal progestin concentrations determined from March 1 to April 30. The percent mink in each group giving birth to live offspring was 42.8%, 80.8% and 92.3% for mink mated once, twice or three times, respectively (P<0.05). Litter sizes did not differ among mink mated once (5.22±0.55), twice (6.29±0.35) or three times (6.08±0.32; P>0.05). Mean fecal progestin concentrations from mating to diapause (March 19) did not differ between mink that whelped or not, nor in response to the number of times mated. However, mean fecal progestin concentrations for mink that whelped were higher on March 25 (peri-implantation) than March 19 after being mated once (51.96±2.96 vs 23.53±1.89nM/g dry wt; P<0.05), twice (66.00±1.60 vs 25.57±1.28nM/g dry wt; P<0.05) or three times (66.48±1.42/g vs 19.16±1.09nM/g dry wt; P<0.05). During implantation (April 5), mean fecal progestin concentrations for mink that whelped after being mated once (146.60±10.02nM/g dry wt), twice (162.10±5.64nM/g dry wt) or three times (188.50±3.92nM/g dry wt) were significantly higher than for those that failed to whelp; 119.30±8.87nM/g dry wt, 77.84±5.86nM/g dry wt. and 118.9±6.55nM/g dry wt., respectively (P<0.05). Our findings suggest that measurement of fecal progestin concentrations during blastocyst reactivation and implantation may be a useful indicator of successful pregnancies in mink.

Identification of orally active, potent, and selective 4-piperazinylquinazolines as antagonists of the platelet-derived growth factor receptor tyrosine kinase family.

We have previously found that the 4-[4-(N-substituted carbamoyl)-1-piperazinyl]-6,7-dimethoxyquinazolines can function as potent and selective inhibitors of platelet-derived growth factor receptor (PDGFR) phosphorylation. A series of highly potent, specific, orally active, small molecule kinase inhibitors directed against members of PDGFR receptor have been developed through modifications of the novel quinazoline template I. Systematic modifications in the A-bicyclic ring and D-rings of protype I were carried out to afford potent analogues, which display IC(50) values of <250 nM in cellular betaPDGFR phosphorylation assays. An optimized analogue in this series, 75 (CT53518), inhibits Flt-3, betaPDGFR, and c-Kit receptor phosphorylation with IC(50) values of 50-200 nM, whereas 15-20-fold less potent activity against CSF-1R was observed. This analogue also inhibits autophosphorylation of Flt-3 ligand-stimulated wild-type Flt-3 and a constitutively activated Flt-3/internal tandem duplication (ITD) with IC(50) values of 30-100 nM. Through this optimization process, 75 was found to be metabolically stable and has desirable pharmacokinetic properties in all animal species studied (F% > 50%, T(1/2) > 8 h). Oral administration of 75 promotes mice survival and significantly delayed disease progression in a Flt-3/ITD-mediated leukemia mouse model and shows efficacy in a nude mouse model of chronic myelomonocytic leukemia.

Discovery of novel 2,8-diazaspiro[4.5]decanes as orally active glycoprotein IIb-IIIa antagonists.

In our efforts to develop orally active GPIIb-IIIa antagonists with improved pharmaceutical properties, we have utilized a novel 2,8-diazaspiro[4.5]decane scaffold as a template. We describe here our investigation of a variety of templates including spiropiperidinyl-gamma-lactams, spiropiperidinylimide, spiropiperidinylureas, and spiropiperidinylhydantoins. With the appropriate acidic and basic pharmacophores in place, each template yielded analogues with potent GPIIb-IIIa inhibitory activity. One of the compounds, 59 (CT50787), was also used to demonstrate for the first time the use of a pharmacological agent which is alphaIIbbeta3 specific to display biological activity in a lower species such as mouse and to extend bleeding times. Evaluation of the pharmacokinetic properties of selected compounds from each series in rat, dog, and cynomolgus monkey has led to the identification of 22 (CT51464), a double prodrug, with excellent pharmacokinetic properties. It exhibited good pharmacokinetic profile across species (F% = 33 (Cyno), 73 (dog), 22 (rat); t(1/2)(beta)() = 14.2 h (Cyno), 8.97 h (dog), 1.81 h (rat)). The biologically active form, 23 (CT50728), displayed inhibition of platelet aggregation in platelet rich plasma (PRP) with an IC(50) value of 53 nM in citrate buffer, 110 nM in PPACK anticoagulated PRP, and 4 nM in solid-phase GPIIb-IIIa competition binding assay (ELISA). Both 23 and 22 were stable in human liver microsomes, did not inhibit the P450 3A4 isozyme, and had low protein binding (18.22% for 23) and a desirable log P (0.45 +/- 0.06 for 22, and -0.91 +/- 0.32 for 23). It is predicted that the high oral bioavailability for these compounds in multiple species should translate into lower intra- and intersubject variability in man. The long plasma half-life of the lead is consistent with once or twice daily administration for chronic therapy. Analogue 22 (CT51464) thus appears to be a promising oral GPIIb-IIIa inhibitor with significantly improved pharmacokinetic properties over the previously described clinical candidates and may be found useful in the treatment of arterial occlusive disorders.

The effects of estradiol and catecholestrogens on uterine glycogen metabolism in mink (Neovison vison).

Glycogen is a uterine histotroph nutrient synthesized by endometrial glands in response to estradiol. The effects of estradiol may be mediated, in part, through the catecholestrogens, 2-hydroxycatecholestradiol (2-OHE2) and 4-hydroxycatecholestradiol (4-OHE2), produced by hydroxylation of estradiol within the endometrium. Using ovariectomized mink, our objectives were to determine the effects of estradiol, 4-OHE2, and 2-OHE2 on uterine: 1) glycogen concentrations and tissue localization; 2) gene expression levels for glycogen synthase, glycogen phosphorylase, and glycogen synthase kinase-3B; and 3) protein expression levels for glycogen synthase kinase-3B (total) and phospho-glycogen synthase kinase-3B (inactive). Whole uterine glycogen concentrations (mean ± SEM, mg/g dry wt) were increased by estradiol (43.79 ± 5.35), 4-OHE2 (48.64 ± 4.02), and 2-OHE2 (41.36 ± 3.23) compared to controls (4.58 ± 1.16; P ≤ 0.05). Percent glycogen content of the glandular epithelia was three-fold greater than the luminal epithelia in response to estradiol and 4-OHE2 (P ≤ 0.05). Expression of glycogen synthase mRNA, the rate limiting enzyme in glycogen synthesis, was increased by 4-OHE2 and 2-OHE2 (P ≤ 0.05), but interestingly, was unaffected by estradiol. Expression of glycogen phosphorylase and glycogen synthase kinase-3B mRNAs were reduced by estradiol, 2-OHE2, and 4-OHE2 (P ≤ 0.05). Uterine phospho-glycogen synthase kinase-3B protein was barely detectable in control mink, whereas all three steroids increased phosphorylation and inactivation of the enzyme (P ≤ 0.05). We concluded that the effects of estradiol on uterine glycogen metabolism were mediated in part through catecholestrogens; perhaps the combined actions of these hormones are required for optimal uterine glycogen synthesis in mink.

Mounting live cells attached to coverslips for microscopy.

INTRODUCTIONThis article describes the mounting of coverslips containing live cells onto microscope slides.

Media for mounting fixed cells on microscope slides.

INTRODUCTIONAfter a specimen is labeled, coverslips containing cells or tissues are mounted onto microscope slides, or slides containing sections are overlaid with a coverslip. A number of recipes for commonly used mounting media are presented in this article, each with particular recommendations.

Hematoxylin and eosin staining of tissue and cell sections.

INTRODUCTIONHematoxylin and eosin (H&E) stains have been used for at least a century and are still essential for recognizing various tissue types and the morphologic changes that form the basis of contemporary cancer diagnosis. The stain has been unchanged for many years because it works well with a variety of fixatives and displays a broad range of cytoplasmic, nuclear, and extracellular matrix features. Hematoxylin has a deep blue-purple color and stains nucleic acids by a complex, incompletely understood reaction. Eosin is pink and stains proteins nonspecifically. In a typical tissue, nuclei are stained blue, whereas the cytoplasm and extracellular matrix have varying degrees of pink staining. Well-fixed cells show considerable intranuclear detail. Nuclei show varying cell-type- and cancer-type-specific patterns of condensation of heterochromatin (hematoxylin staining) that are diagnostically very important. Nucleoli stain with eosin. If abundant polyribosomes are present, the cytoplasm will have a distinct blue cast. The Golgi zone can be tentatively identified by the absence of staining in a region next to the nucleus. Thus, the stain discloses abundant structural information, with specific functional implications. A limitation of hematoxylin staining is that it is incompatible with immunofluorescence. It is useful, however, to stain one serial paraffin section from a tissue in which immunofluorescence will be performed. Hematoxylin, generally without eosin, is useful as a counterstain for many immunohistochemical or hybridization procedures that use colorimetric substrates (such as alkaline phosphatase or peroxidase). This protocol describes H&E staining of tissue and cell sections.

Cutting sections of paraffin-embedded tissues.

INTRODUCTIONThis protocol describes the sectioning of tissues embedded in paraffin blocks. Paraffin sections require extensive fixation and processing steps but provide superior morphology compared with other sectioning methods. Sectioning paraffin blocks requires experience and should be learned from an experienced researcher, if possible.

Preparation of slides and coverslips for microscopy.

INTRODUCTIONIt is imperative that the slides and coverslips used in fluorescence microscopy procedures be extremely clean. Although coverslips look clean, especially when a new box is first opened, they may have a thin film of grease on them that will not allow tissue culture cells to adhere well and that may interfere with some processing steps in certain protocols. Therefore, coverslips should routinely be washed with acid or base solutions to rid them of this film. Commercial precleaned slides are also likely to be dirty and must be washed prior to use. This protocol describes various approaches for cleaning slides and coverslips and sterilizing them for cell culture, as well as methods for subbing slides. In the subbing procedure, slides are coated with gelatin, aminoalkylsilane, or poly-L-lysine solution to promote the adhesion of cells or tissues to the glass surface. Gelatin or aminoalkylsilane is usually used for tissue sections or small organisms, whereas poly-L-lysine is routinely used for cultured cells.

Paraffin embedding tissue samples for sectioning.

INTRODUCTIONThis protocol describes a method for embedding tissues in paraffin blocks for sectioning. Paraffin sections require extensive fixation and processing steps, but provide superior morphology compared with other sectioning methods.

Decalcifying tissues for paraffin embedding.

INTRODUCTIONParaffin sections of bone usually require a decalcification step after fixation before sectioning. This protocol describes a method for decalcifying fixed tissue.