Utilizing a variety of experimental approaches, as well as knockdown of beclin , and treatment method with methyladenine , a regarded inhibitor of autophagy in mammalian cells, we have been not able to inhibit non apoptotic cell death induced by saquinavir . Our get the job done demonstrates that endoplasmic reticulum strain and autophagy are an essential mechanism of protease inhibitor mediated cell death in ovarian cancer cells. In conclusion, the capability to restore or circumvent apoptotic cell death pathways is central for the growth of novel therapies for ovarian cancer, provided that defective apoptosis underlies the chemoresistance that develops and limits successful treatment for individuals . Raising interest and efforts are focused on therapeutic approaches focusing on autophagy . Our job not just highlights a whole new class of medicines that triggers ovarian cancer cell death, but it also demonstrates the capacity of protease inhibitors to induce cell death in ovarian cancer cells that happen to be resistant to standard chemotherapy. Protease inhibitors are FDA accredited by using a good safety profile that permits their clinical use.
The syk kinase inhibitors means of saquinavir to induce caspase dependent apoptosis at the same time as caspase independent endoplasmic reticulum worry and autophagy helps make it a wonderful therapeutic agent for continued investigation. A lately published phase I clinical trial implementing the protease inhibitor nelfinavir in patients with locally state-of-the-art pancreatic cancer demonstrated acceptable toxicity and promising anti tumor activity . Given these findings, protease inhibitors which include saquinavir warrant additional investigation each in an in vivo tumor model of ovarian cancer and in the end in clinical trials in patients with ovarian cancer. Recurrence and subsequent acquired chemoresistance are accountable for that therapeutic failure occurring in about of ovarian carcinoma circumstances. This poor prognosis areas ovarian carcinoma since the main result in of death by gynecological malignancy, regardless of the advances in chemotherapy during the last decades.
Traditional remedy of ovarian cancer contains debulking surgery and subsequent platinum based chemotherapy, by which cisplatin or carboplatin is generally related with cyclophosphamide or paclitaxel . A number of mechanisms can contribute to cisplatin resistance in tumor selleck chemicals SB 415286 cells, as well as decreased intracellular drug accumulation , enhanced detoxification , increased DNA restore , tolerance in direction of platinum adducts and DNA hypermethylation . Considering cisplatin and the majority of chemotherapeutic agents exert their cytotoxic effect on tumor cells by inducing apoptotic cell death consequently of lethal DNA damage , a decreased susceptibility to apoptosis as a consequence of defects during the apoptotic or survival pathways has also been held accountable for chemoresistance .

The elaboration of new biochemical resources can be necessary to acquire a direct demonstration, but the present final results lead to a scheme where compartmentalization is important for the sequence of events. CPEB, unphosphorylated in G prophase blocked oocytes, is subjected towards the antagonistic action of cdc kinase and of PP soon after hormone addition. A single can assume that just before nuclear envelope breakdown, localized PP exercise is dominant and that an Inh like inhibitor is sequestered from the nucleus. On the G to M phase transition, improvements in nucleocytoplasmic visitors and nuclear envelope integrity makes it possible for the release within the inhibitor from the cytoplasm. The resulting inhibition of PP will allow full phosphorylation of CPEB and also the ensuing polyadenylation, then translation of cyclin B mRNAs. This scheme could possibly be an oversimplification seeing that over one particular phase, leading to cyclin B synthesis, might possibly be managed through the Inh delicate phosphatase. Aurora continues to be this kind of a feasible mediator, regardless of the lack of evidence stated above, as the significance of its activation remains for being elucidated.
The neural crest is usually a specialized population of cells that develops on the border amongst the neural plate and the epidermis in all vertebrate embryos. The closure in the neural tube brings these cells together with the dorsal pole on the neural tube, from in which they migrate to several areas with the embryo, and differentiate into a number of cell forms . From research in chick, amphibian and zebrafish embryos, a number of the signals involved in the induction from the neural crest are recognized, selleck chemical ZM306416 e.g BMPs, Wnts, FGF, Notch and retinoic acid . In Xenopus and zebrafish, it appears the initial induction with the neural crest depends on a gradient of BMP action . As this kind of, the intermediate concentrations of BMPs located on the border amongst the neural plate as well as epidermis are individuals required to specify the neural crest cells, i.e the place BMP exercise is lower than that demanded to induce epidermis formation and higher than that which induces neural tissue .
It can be now accepted that Apixaban the induction of your neural crest may be a multistep process, whereby a precise sequence of signals is critical to provide the exact number of crest cells with the best position. Even so, it’s clear the amount of cells within a provided organ or tissue can be established from the fee of cell division and of cell death . Thus, after inductive signals are already transmitted to a population of cells, supplemental mechanisms are expected to find out the precise size with the organ by controlling cell proliferation and death. Indeed, throughout animal growth, various structures are formed that are later on eliminated by apoptosis.

All through early advancement, the proper spatiotemporal expression of nodal ligands and also the tight regulation of signaling amounts are very important for specification of mesendoderm and for left proper patterning . Inside the frog, Alk appears to get the form I receptor accountable for that early nodal signaling demanded for right patterning of your germ layers while in gastrulation as well as for left ideal patterning through tailbud phases. From the mouse, reduction of Alk effects in early and lethal defects in egg cylinder organization and gastrulation . In vitro evidence suggests that Alk may perhaps have the ability to respond to nodal signals in some contexts, but an Alk knockout has no phenotype . Alk would seem for being responsible for later on developmental occasions, such as heart patterning and angiogenesis . Whilst very much has become discovered by way of loss of function evaluation about the part of activin nodal ligands and their receptors in development, you will discover in lots of cases technical limitations to your interpretation of this examination.
To start with, late developmental phenotypes are often obscured by disruptions in signaling during early improvement, and second, signaling elements might be partially or entirely redundant, generating unclear the perform of personal elements. Even though tissuespecific gene inactivation while in the mouse using the Cre lox process has additional resources provided an strategy to the very first of those challenges, it can be limited by the temporal and spatial resolution of gene expression driven by accessible enhancers, does not effortlessly handle the challenge of practical redundancy, and is not currently applicable to other embryological model organisms. Chemical inhibitors, that are easily applied to anamniote embryos, may be readily added and removed and can concurrently inactivate closely associated and partially redundant signaling components, providing a possibly helpful complement to tissue specific gene inactivation within the examine of temporally particular roles of developmental signals. A chemical inhibitor of activin nodal signaling, SB , inhibits Alk kinase activity in vitro with an IC of nM, and in addition inhibits Alk and Alk with related potency .
Studies in cell culture have proven that SB can inhibit the two Smad phosphorylation and downstream reporter gene expression . Whilst inhibitors like SB are probably useful probes of activin nodal signaling perform while in embryogenesis, a significant concern concerning the use of purchase SB590885 such inhibitors is their specificity in vivo. A molecule which is made to bind from the active web page of the unique protein might possibly also bind and have an impact on other structurally associated but functionally distinct proteins. This is certainly of unique concern for minor molecules targeted to ATP binding sites similar to SB , due to the fact in vitro specificity scientific studies can by no means entirely handle the result with the inhibitor around the complete assortment of nucleotide binding proteins current in vivo.

In most species, VEGF mRNA is detected in the granulosa derived luteal cells from the newly formed corpus luteum, and VEGF protein is localized in steroidogenic cells in the corpus luteum . Autocrine or paracrine results of luteal prostaglandins might be involved in the handle in the corpus luteum lifespan and functions . Our earlier study demonstrated that cyclooxygenase exercise might be necessary for angiogenesis from the early building corpus luteum in rats . When gonadotropinprimed rats were injected by using a COX inhibitor, NS , for days right after ovulation, serum progesterone levels decreased with each other with vasculature impairment from the creating corpus luteum. VEGF stimulates the expressions of COX and prostaglandin E synthase mRNAs in rat luteal cells . Hence, COX might be involved in the physiological angiogenesis with the corpus luteum that will take place through the early luteal phase in rats. To handle the part of eicosanoids in luteal angiogenesis in the current examine, we examined the effects of exogenous PGE, Ciprostene , and U on progesterone secretion and angiogenesis making use of the gonadotropin induced ovulation model in rats.
We also examined the feasible part of eicosanoids in VEGFinduced ovarian angiogenesis Resources and systems Animals Immature Wistar Imamichi rats had been maintained in an air conditioned area underneath managed lighting with totally free accessibility to meals and water. All experimental protocols with rats utilized in this review were reviewed and accepted by the Institutional Animal Care Committees rho kinase inhibitors with the Tokyo University of Pharmacy and Life Science, in compliance with institutional tips for experimental animal care. Pseudopregnancy was induced by substantial dose gonadotropin, equine chorionic gonadotropin , and human CG administered h following eCG therapy Experimental schedule Gonadotropin primed rats have been injected with NS , a selective COX inhibitor, at h to the day of ovulation along with the following day . The dose of NS enough to inhibit COX exercise was determined in our prior research . At h on day , rats were anesthetized with ether, the uterus was exposed, the middle on the uterine horn was ligated, and also the ovary about the side opposite of the place the injection would arise was eliminated.
PGE , Ciprostene , or U was injected into the ligated uterine lumen close to the ovary. The doses of eicosanoids utilized have been established primarily based on the physiological contents from the preovulatory ovary reported by Brown and Poyser . The mixture of those 3 eicosanoids was also administered as a remedy. Animals from the manage group were injected with all the same quantity of PBS containing Semagacestat . gelatin like a car. Following the injections, animals had been sutured with silk thread. Two days later , animals have been anesthetized with ether and decapitated, and their ovaries have been straight away eliminated to measure hemoglobin written content.

The proteins have been then visualized making use of an enhanced chemiluminescence detection kit siRNA transfection To knockdownATM expression, synthetic ATMsiRNA duplex oligomer as well as a scrambled siRNA duplex oligomer were purchased from Utilized Biosystems. For siRNA transfection experiments, A cells had been plated onto mmdishes and cultured overnight in full medium. The following morning, cells have been transiently transfected with Oligofectamine supplemented with ATM siRNA . At h submit transfection, cells have been treated with or not having emodin for an alternative h. Cells were then harvested for detection the protein level of ATM by immunoblotting Complete RNA planning and RT PCR analysis The cells have been trypsinized and washed twice with PBS. Total RNA was ready using a Qiagen RNA extraction kit. The RNA concentration was established by studying the absorbance at and nm using a UV spectrophotometer. A total of g of cDNA was synthesized as outlined by the manufacturer’s instructions . For PCR amplification of specific genes, a response mixture that contained M dNTP, mM MgCl, pmol primers, unit of Taq polymerase and g of cDNA item was prepared on ice.
PCR was performed at the exponential selection, plus the PCR goods had been separated by electrophoresis on agarose gels stained with ethidium bromide and analyzed applying the Ever Gene Image Process. actin gene was analyzed as an internal loading handle. The measurement of mitochondrial membrane potentials and reactive oxygen species generation were performed as previously described . Briefly, A cells were treated selleck Secretase inhibitors with or while not M emodin at the indicated time points. Soon after therapy, the cells had been incubated with , dichlorodihydrofluorescein diacetate , dihydroethidine or JC at C for a further min. The cells had been then washed 3 times with a cold PBS solution, along with the fluorescence intensity on the cells was analyzed utilizing a Becton Dickinson Flow cytometer. Statistical examination Each of the figures proven in this articlewere obtained fromat least three independent experimentswith very similar success. All information are presented as imply S.E.M. of a minimum of three separate experiments. Statistical differences had been evaluated using the Student’s t check and considered major at P P .
or P . Outcomes Emodin treatment method induces phosphatase inhibitor library the accumulation of p and Bax, but decreases the expression of survivin We previously demonstrated that emodin could selectively kill human lung adenocarcinoma A cells, but not non tumor cells including human fibroblast like lung WI cells, by activating a reactive oxygen species dependent mitochondrial pathway . Furthermore, emodin exerts anti tumorigenic activity by inducing apoptosis in many cancer cells. Because emodin has become demonstrated to become a genotoxic compound, and due to the fact most cytotoxic drugs induce apoptosis by activating the p dependent pathway, we investigated no matter if p plays a part in emodin triggered apoptosis in human lung adenocarcinoma A cells.

Because cells in RIPA buffer supplemented with . Triton X . Following incubation for min at ice bath temperature, cells were sonicated for s in ice bath and centrifuged at , g for min at C. Aliquots in the cell extracts were taken for protein determination. Pre cleared cell extracts containing g of protein have been incubated with rabbit polyclonal anti IGF I receptor subunit antibody overnight at C with constant rocking. Following the addition of l of Protein G Plus Protein A agarose slurry , the samples have been even further incubated at C for h. The beads were then washed four occasions with RIPA buffer and mixed with electrophoresis sample buffer Animals and tissue dissections Male Sprague Dawley rats andmale CD mice weremaintained within a h light dark cyclewith food andwater ad libitum. Experiments had been carried out in accordance together with the European Communities Council Directive of November and together with the concepts of Laboratory Animal Care in Italy .
Nucleus accumbens was isolated from m thick coronal brain slices by microdissection as previously described Tissue slice treatment method Nucleus accumbens slices were incubated within a freshly oxygenated Krebs Hepes buffer containing NU7441 mM Hepes NaOH, mM glucose, mM NaCl, mM KCl mM MgSO mM CaCl mMKHPO at C for min. Thereafter, the tissue slices had been incubated in the similar buffer at C and exposed to both naltrindole or car for min, followed by min publicity to NDMC or motor vehicle. The medium was aspirated and l of ice cold RIPA buffer was added. The samples were homogenized by sonication and stored at ? C In vivo treatment method CD mice were handled with naltrindole or saline min just before the administration of either vehicle or NDMC . NDMC was dissolved in glacial acetic acid as well as the choice was buffered to pH The animals had been sacrificed by cervical dislocation min just after NDMC administration. The brain was quickly eliminated and nucleus accumbens was dissected from brain coronal slices as indicated above. Tissue from every single animal was collected in l of ice cold RIPA buffer, sonicated and stored at ? C.
Aliquots within the tissue extracts have been taken for protein determination Western blot analysis Aliquots of cell or tissue extracts containing equal volume of protein had been subjected to SDS polyacrylamide gel electrophoresis and the proteins were electrophoretically Rutaecarpine transferred to polyvinylidene difluoride membranes . The efficiency in the transfer was controlled by gel staining and by following the transfer of pre stained protein requirements. Nonspecific binding online websites were blocked by incubation in mM Tris HCl, mM NaCl and . Tween containing BSA for h. Following washing with TBS T buffer, the membranes were incubated overnight at C with 1 within the following antibodies: anti phospho Ser GSK , anti phospho Thr Akt , anti GSK , anti Akt anti phospho IGF I receptor insulin receptor , anti IGF I receptor and anti phosphotyrosine .

Cofilin certainly is the key regulator required for actin polymerization depolymerization. Cofilin contributes to directional motility of cells , hence, could have very important implications towards the motility and invasion of cancer cells. There’s no direct evidence to recommend that the CD induces variety of metastatic susceptible cells. Our earlier operate has proven the variant CD v isoform modulates cofilin expression and phosphorylation . On this research, we applied human colon cancer cells that tremendously express CD, stable cell lines created in which CD was inhibited by siRNA, and murine colon from CD knockout and wild sort mice, to examine the purpose of CD in modulating cell migration. Materials and techniques siRNA siRNAs were synthesized by Dharmacon Inc siRNA sequences targeting human CD was put to use on this review. Antibodies Mouse anti human CD , mouse anti human actin , rabbit anti Lyn and CD , rabbit anti phosphorylated AKT and complete AKT , rabbit anti cofilin have been implemented in the Western blot and immunohistochemistry studies. Secondary antibodies were goat anti mouse and anti rabbit FITC , goat anti rabbit HRP and goat anti mouse HRP .
Cell culture HT and SW human colon cancer cells were grown working with DMEM medium containing FBS with penicillin streptomycin antibiotics . SW transfected with CD isoforms , v and v isoform and empty vector construct have been maintained as described . Optimem, oligofectamine, glutamine, penicillin and streptomycin had been obtained from SB 525334 price Invitrogen. Mice CD ? ? mice had been obtained from Dr. Tak Mak . Age and sex matched wild type mice had been obtained from Jackson Laboratory . In vitro transfection of HT cells with siRNA For stable transfection, HT cells were trypsinized and resuspended in fresh DMEM medium without having antibiotics. Cells had been plated in very well plates each day just before transfection, corresponding to a density of on the time of transfection and transfected with lipofectamine and plasmid DNA vector that has the CD siRNA insert as per the producer?s protocol with g ml of hygromycin because the antibiotic of option for that selection of secure clones.
Following the choice operation, the clones were maintained in serum medium containing g ml of hygromycin. Detection of siRNA mediated gene silencing by Western selleck chemicals TKI-258 blot analysis Cell lysates had been prepared and Western blotting carried out as described . Briefly, the cellswere lysed in lysis buffer containing the cocktail of protease inhibitors . Concentrated Laemmli sodium dodecyl sulfate sample bufferwas added on the cell lysates and incubated for min within a boiling water bath, vortexed and proper sum loaded onto a SDS PAGEforWestern blot analysiswith appropriate main antibody for CD. Western blot examination as outlined above was similarly performed for AKT phosphorylation , total AKT, cofilin, and Lyn kinase. Representative Western blots from one of at the least 3 comparable experiments are proven.

Also, the data to get presented will argue for inhibition of the phase besides the EGFR since the significant point of make contact with inhibition of EGF dependent development. Examination of EGF dependent Akt activation EGF dependent Akt activation was examined to find out if suppression in the EGFR in higher density cells has any effect on downstream EGF dependent pathways. The phosphorylation specified Akt antibody, phosphoserine , was applied to assess Akt activation . In contrast on the decreased EGFR activation observed at all time points during the large density cells, EGF similarly activated Akt at min and min in each large and minimal density cells. Soon after min, in contrast towards the minimal density cells, Akt activation markedly decreased by while in the highdensity cells . Akt activation remained rather continuous throughout the min time program inside the minimal density cells . The mass of Akt was comparable underneath each density conditions , and h catenin showed no variation beneath the higher and lower density problems . These final results indicate that Akt activation at the same time as EGFR activation in substantial density cells was decreased, however the time program of suppression of Akt and EGFR actions differ.
At this time in our experiments, it was unclear when the suppressed EGF dependent Akt activation from the high density cells was only a direct reflection from the decreased EGFR activation in these cells or if large density right suppresses EGF dependent Akt activation. The remainder of mGlur3 agonist our experiments will show a brand new paradigm for speak to inhibition of development, that direct suppression of Akt activation instead of the suppressed EGFR activation is liable for contact inhibition of EGF dependent growth of these cells. Analysis of EGF dependent Erk activation EGFR activation is suppressed in large density cells relative to lower density cells; it would be predicted that all EGF dependent signals downstream of the EGFR must be inhibited relative on the reduced density cells. To check this hypothesis, EGF dependent Erk activation was examined. As noticed in Fig. A, Erk was activated during the substantial density cells although the EGFR in these cells had been less activated , and Erk was activated to very similar extents from the highand very low density cells.
Similarly, EGF dependent Erk activation from the substantial density cells was much like the lowdensity cells . Erk Cisplatin masses had been related at both densities . Examination in the EGFR substrates, Gab and erbB, and their capability to activate PI kinase While suppressed relative to very low density cells, the magnitude of EGFR activation in large density cells appears enough to totally activate the EGF dependent Erk pathway . Why does density dependent suppression of EGFR activity depart the EGF dependent Erk pathway unaffected though suppressing the EGF dependent Akt pathway? We examined the tyrosine phosphorylation states of EGFR substrates involved in Akt activation, Gab and erbB, to start to reply that question.

Elements and methods Chemical compounds Geldanamycin was obtained from Invivogen and dissolved in DMSO. The PI kinase inhibitor LY and cycloheximide were obtained from Sigma Aldrich and dissolved in DMSO and water respectively. Calyculin A, a phosphatase inhibitor, was bought from Cell Signaling. Cell culture Murine hematopoietic Ba F cells were maintained in RPMI medium supplemented with heat inactivated fetal calf serum and ng ml mouse recombinant IL . Ba F cells stably transfected together with the MSCV retroviral vector have been cultured from the previously described medium with all the addition of mg ml G . The SR cell line was cultured in RPMI with FCS. Every one of the cell lines had been incubated at C in CO and had been passaged after they reached a density of approximately . to ml. Twentyfour hrs prior to therapies the cells have been transferred in medium not having antibiotics. To the experiments shown in Fig the phosphatase inhibitor Calyculin A was additional to a final concentration of nM min just before cell harvesting. For that isolation of bone marrow cells, healthier BALB c mice were sacrificed by CO asphyxiation followed by cervical dislocation.
Bone marrow cells have been isolated by flushing femurs and tibias with ice cold PBS and cultured in RPMI with FCS. Viability and development curve examination Cell viability more info here was assayed by the trypan blue exclusion procedure. Development curves soon after geldanamycin or LY remedies have been conducted employing the CellTiter Glo? Luminescent Assay of Promega in line with the manufacturer’s directions. Western blotting and antibodies For every sample, cells had been collected by centrifugation , washed when with ice cold PBS and lysed in l of lysis buffer containing SDS, mM HEPES M NaCl, mM EDTA, glycerol, mM glycerophosphate, mM phenylmethylsulfonyl fluoride, mM NaF and protease and phosphatase inhibitors . Protein concentration was determined implementing the BCA reagent . Samples of g have been analyzed in SDS polyacrylamide gels, transferred to PVDF membranes and blocked for h at space temperature with nonfat dry milk in TBS buffer . Incubation together with the key antibodies was completed at area temperature for h or overnight at C.
Following three washes with TBS supplemented with . Tween the membranes were incubated with all the proper secondary antibody for h at area temperature. Following three far more selleck chemical phosphatase inhibitor library washes the blots have been handled with the enhanced chemiluminescence reagent and exposed to x ray film for detection. Also,Western blots were quantified utilizing a Licor Odyssey Infrared imaging technique. Antibodies utilized have been: Akt, Akt , Cdk , Cdc, Hsp and Hsp . Secondary antibodies for use with the Licor system were IRDye CW conjugated goat anti rabbit and IRDye conjugated goat anti mouse. cells treated with DMSO or geldanamycin have been lysed in l of Nonidet P lysis buffer . Cell lysates have been cleared by centrifugation at C for min and l of your extract was utilised for protein quantification from the Bradford assay.

Overall, these Aurora kinase inhibitors don’t end cells from getting into mitosis but cause defects in chromosome segregation . Although cells exposed to Aurora kinase inhibitors exit mitosis, they’re not able to divide, a phenotype related most closely with inhibition of Aurora B . Human tumor cells are prone to killing by Aurora kinase inhibitors, nevertheless the mechanism of killing is not really thoroughly understood. Given that these medication block cell division, continued progression via the cell cycle can build polyploid cells that could undergo apoptosis. Some scientific studies have implicated p from the response to Aurora kinase inhibitors . Cells lacking p showan enhanced means to rereplicate DNA when cytokinesis is blocked by Aurora kinase inhibitors. Our investigation was aimed at evaluating the effects of Aurora kinase inhibitors in isogenic pairs of cells that only differed in p standing. We observed that although p did decelerate cell cycle progression right after remedy with both ZM or VE , this cell cycle block was not wholly penetrant. Induction with the p response is correlated with the physical appearance of localized DNA damage just after inhibition of Aurora kinases.
Elimination of you can check here the drug immediately after several days permitted some cells to evade killing through the Aurora kinase inhibitor. These clones have been not resistant to the drug on re publicity and normally showed alterations in ploidy. The origin of some of these colonies could possibly involve the asymmetric division of multinucleated giant cells. Strategies Cell lines and culture problems Parental HCT cells, initially derived from a human colon carcinoma, have wild form p and had been in contrast to HCT cells in which the two p alleles had been inactivated by homologous recombination . The HT cell line was initially derived from a human fibrosarcoma and has wild type p. HT GSE cells have been developed by infecting that has a retrovirus expressing GSE , a dominant unfavorable edition of p. The HT LXSN cell line was infected with all the empty retrovirus vector . The HelaM cell line is usually a subclone within the Hela cervical carcinoma cell line . Cells were grown in plastic culture plates in an environment of C and CO.
All cells had been grown in Dulbecco’s minimum necessary medium supplemented with fetal bovine serum , penicillin , and streptomycin . ZM and VE were dissolved in dimethyl sulfoxide and had been obtained from Astra Zeneca Pharmaceuticals Ltd. and Merck Co respectively. Caffeine was dissolved in Dulbecco’s minimum important medium. To induce DNA harm, cells had been treated with Adriamycin at . g ml or Etoposide at M. Colonies have been visualized ZD-1839 by staining with a saturated solution of methylene blue in ethanol. Flow cytometry Cells removed from plates implementing trypsin have been mixed with floating cells in the tissue culture medium, and all cells have been collected by centrifugation at g, for min at C. The cells have been resuspended in phosphate buffered saline and fixed in ethanol at ? C for not less than h .