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It’s titled, “An Unfaithful Husband.” I need someone to play the husband. I’ll ask Lee Kichan to play it since he acted in Hollywood. Let’s see what Hollywood acting is like. (Shy) Hello. I’m a woman in my 30s with three daughters. I’m busy raising our kids and doing house chores. But this one burden is frustrating me. Ring. Ring. “Honey, where are you? Why didn’t you answer your phone?” In English. “Why are you calling so much? I’m here in front of Mokpo Sea.” How would you say this? (Perfect to the end) See you later. My husband who travels all over the country is crazy about sea fishing. Sea fishing? And he’s so brazen. “My fishing rod broke. Go get it fixed. Be careful with it when you send it in.” He even makes me run errands. The problem is that in my womb is our fourth child. This guy is unbelievable. “Honey.” Wait. I thought you played the role well until now. But it sounds like he’s worse than we thought. She’s pregnant. This is ridiculous. You must be meaner. “Honey, we have to buy the baby’s things now.” “Right! I need to buy another fishing rod. I have to buy a reel, too. What else?” (The audience is satisfied) That really sounded mean. “Honey, I must go in for a checkup.” “I can’t go with you!” That’s right. – “I’m tired.” / – Good. His eyes sparkle when he goes fishing. Why can’t he see his soon-to-be-born baby? If he continues to be like this I can’t stand to live with him anymore. Please help me knock some sense into him. This is her concern. This is unbelievable. Tell me what you thought about Kichan’s acting. I can’t go with you! How scary. I can’t go with you. I must go fishing. – Go back to your seat. / – Okay. (He’s back to his innocent self) Let’s bring her out now. (Who’s concerned about her husband?) (Cheon Yujeong) (She’s close to her due date) Minkyoung, stand up. We know how she feels. – Sure. / – Sure. It’s hard to get up, right? (For different reasons) Will you be all right? To be honest, it’s hard. – Right now? / – Yes. My pelvis hurts a lot. My pelvis has been hurting since I got pregnant. You must’ve been very upset to come here today. It’s hard enough just to rest at home. I really felt like I was going to die. You’re pregnant with your fourth child. How long has this been going on? For 8 years. – Like this? / – Yes. – He goes fishing and doesn’t help you? / – Right. (No way) He wasn’t like this when we got married. After our eldest was born he went fishing with his brother and got into sea fishing. He got into after he went with his brother. – You must hate his brother. / – Yes. She answered right away. How often does he go fishing? He’s out of his mind! He’s out of his mind? I understand. He goes every day. He goes whenever he doesn’t work. Not on weekdays, right? He goes on weekdays and on weekends. When is he home then? He’s never home when he’s off work. He loves sea fishing so much that he travels to Gangwon-do, Tae-an, Mokpo… He travels all over the country. He said he’d be back soon and was gone for 6 days. What? (Soon means 6 days) – “I’ll be right back.” / – He’s quite different. Did he go overseas? No. He went to Haenam. And then he yelled at me. Why? “I went to have fun. What’s wrong with that?” He didn’t apologize? No. I never heard him apologize. – Never? / – Never. He still gets angry when I talk about it. (Wow, that’s worrisome) If he goes that often it must’ve cost a lot of money. He’s gone fishing for 8 years now and has spent more than $10,000. He has 6 fishing rods in the balcony, 8 reels and 3 at his parents’ home. He has to pay for the boat when he goes out. He has to pay for gas for his trip, too. He spends $200-300 on each trip. About $200-300. I told him once that we need to buy baby supplies. He said, “I need to buy another fishing rod and a reel.” He didn’t even listen to you? He just ended the conversation there. (So frustrating) If you decided to come here you must’ve tried to talk to him many times. I did, but all he said was, “Okay.” That’s it? I suggested that we come here today. I told him that I sent in my concern. He said, “Let’s see who wins.” He’s that confident about himself? Yeah. Let’s see who wins. (Eager to fight) I’ve been watching this program for years. Many people think that it’s fake. I wondered how people can come out here. Right. I thought I’d be too embarrassed to come. Now I get it. They don’t know what they’re doing is wrong. That’s why he’s so confident. Let’s let him know. I realize now that this is for real. If what she’s saying is true, her husband would be crushed. He can’t have a social life anymore. I hope he has a good reason. Let’s meet her husband. Where are you? Hello. They’re applauding because you’re here today. (Brace yourself) You’re here because you have something to say? Yes. Let’s hear you out now. At first, I didn’t think this was a concern. There’s a big age difference between us. She’s 10 years younger than I am, so I thought maybe it’s because she’s too young. You think it’s because of the age difference? I did try my best. I used to go fishing 4-5 times a week. But after she got pregnant, I go maybe once a week. I don’t go as often anymore. Do you look after your kids when you’re home? Yes. I bathe them when I’m at home. I’ve been doing it for years. – Even now? / – Yes. But recently, my back started to hurt, so I haven’t been washing them for about 4 months. She doesn’t remember the good things. She only remembers the bad things. The sword we took out became dull. He says he bathed the kids every evening. He bathed all 3 kids. Like I said, my pelvis hurts very badly. – I can’t lie down either. / – That’s hard. So I had to ask him to wash them for me. That is why. But he thinks he did all the work. – He’s boasting about it? / – Yes. – Like he’s always done it? / – Yes. (A shy smile) Is it true that you asked her to get your fishing rod fixed? Yes, it’s true. I have to send it in to a certain company. If I ask my wife to do it, she does a good, thorough job. (How frustrating) You ignored her concerns about baby supplies? Baby supplies? – Did you hear it? / – You don’t remember? Of course he heard it. (Guilty) So did someone come to pick the rod up? No, I had to take it with me. – To the post office. / – To the post office? – In your condition? / – With 3 kids. With all 3 kids? (What?) How? I hold two kids and one walks by herself. – One walks by herself? / – Yes. Then you yell, “Watch out for the cars!” Come back here! “Watch out for the red light!” Like this? How did you carry the rod? In my hand. Or I carried it in a bag. A bag on your shoulder, two kids in your hands. Yelling at one kid. – That’s how it goes. / – What else? He always calls when he goes on a business trip. What does he say? “Yujeong, send me my fishing rod.” Oh, he goes on business trips. I told him that I couldn’t do it. – He got upset. / – He did? He got mad and hung up on me. He’s terrible in so many ways. You could’ve rented the equipment. Did you have to ask your wife to send it to you? There was no rental place. That’s not true. Even for gamers, some people won’t play unless they have their own keyboard and mouse. Some people must have their equipment. They even bring their own to internet cafes. I think he’s the type to use his own at all times. Is that right? Yes, it’s right. (I don’t believe this…) It must cost a lot to go fishing. How much do you spend each month? About $500-600. Since it costs $500 each time, that’s $2500 for a month, right? – I guess so. / – That’s what I think. Do you handle your family’s finances? No, I don’t. – Then who does? / – My husband does. – How do you not know? / – Since when? Since we got married. I get an allowance from him for living expenses. I’m afraid to spend money because of it. One time, I bought hairpins for our girls. Their old ones were too old. I didn’t spend hundreds of dollars. Just $2-3 for each one. She’s getting emotional. – $2-3 per hairpin. / – How sad. He said, “You could’ve made them. Why did you spend money?” Make their hairpins? You should make your own fishing rod. Just snap a branch and use that as a rod. It’s the same thing. He has never bought me a birthday gift. – Even when you were dating? / – No. All he did was cook seaweed soup for me. What was so great about him? I was blinded by love. What about when you were pregnant? He bought things only when I begged him. Like clothes. My stomach is quite big this time. I asked my husband for money and I bought shirts for $5 or $10. But he buys brand-name shoes and clothes for himself. He has everything. They are all expensive. Whenever I spend money I have to call and tell my husband. “I bought this and spent this much.” I have to tell him every time. Even now? If I don’t, he yells at me. Let me ask you this. Why do you buy expensive clothes for yourself, but yell at her for spending a few bucks? She’s really good at making things. What? (He’s suggesting that she makes her own things) (Must I put up with this?) She’s good with her hands. I want to sew his mouth shut. It makes me so angry. I think everybody here might vote against you. I’ll give you a chance to explain. You never got her a birthday gift. Not even when you were dating. Why is that? I made her seaweed soup each year. Mom can make seaweed soup, too. She’s not living with her mother, is she? It never occurred to you to buy her a gift? I once asked her what she wants for her birthday. I told her I would buy her anything she wants. She told me she didn’t need anything, just a bowl of seaweed soup. – She said that? / – Yes. She was being considerate. You obey her quite well. That’s right. When she’s pregnant, she needs maternity clothes. You know that, right? She asked for money this time to buy some. You gave her $5? This is the only time he gave me money. What about during your first pregnancy? My mother bought me two maternity outfits. I wore the same clothes for our second child. Also for our third child. They don’t fit this time, so I asked him for money and he gave me $10. I bought it at an underground store. They sell $5 maternity clothes? Yes, just a simple, big shirt. Minkyoung, take off your clothes. I’ll give my outfit to her after the shoot. (Please calm down) I will. I can take my clothes off for her. This is so sad. Unbelievable. Since you’re here today, let’s get it all out. This is why your wife is frustrated. You have to be consistent. Then she would understand that you’re frugal. She wouldn’t be disappointed. That’s what she’s trying to say. I don’t buy new clothes that often. I only buy once or twice a year. When I do buy something, I buy something nice. How much did your shoes cost? My sneakers? About $150? What about your shirts? $150-200 on average. (Disappointed) Husbands care a lot during the first pregnancy. Right. They help out their wives a lot. How was he during your first pregnancy? I really craved fermented skate fish. So I asked him to buy some for me. He said, “Okay, okay.” I begged him for 2 months. “I want to eat fermented skate. Buy some for me. Please.” He finally bought it when his friends came over and had a drink at our place. This time, I really wanted to eat duck meat. That was more than 2 months ago. He still hasn’t bought me grilled duck meat. A friend of mine felt so bad for me, and said, “Let’s go eat duck meat.” I ate ducks until my stomach was about to burst. Multiple ducks… – Because someone else paid for it? / – Yes. I’m really curious. This doesn’t have to be aired. Why do you do that? It’s so hard to understand for us. She had to keep begging you to get her something. She’s your wife, not a stranger. When I got home it was around 11 p.m. The skate restaurant was closed that day. There are many places that deliver. No, we only eat at that restaurant. They serve this certain skate dish. Only that place has good fermented skate. You only eat fermented skate from that place? At the time, I didn’t really care. I just really wanted fermented skate. Now that we’ve talked to your husband for a while I can’t help but feel incredibly frustrated. He makes all kinds of excuses in a calm voice, so as I listen to him… I’d say, “Fine! Forget it! I don’t want it anymore!” – What do you think? / – He makes you give up. It feels like something is stuffed inside me. He frustrates me so much. If I had something sharp, I’d like to poke him. With something long. (Please do it for me, too) – He’s so annoying. / – Fishing. Two months ago, we had an argument and I got a cramp. I couldn’t move at all. So I called an ambulance. I was getting an IV and he said, “You deserved it.” “You deserved it.” – Why? / – Because I talked back to him. Because I didn’t listen to him. (I give up) You’re the worst person I’ve met this year. No, in my lifetime. (Getting more subscribers) He never gets this upset. I tend to joke around like that. No, please don’t say that. Please don’t say that you were kidding. Maybe he meant to say something else. Something like, “You preserved it.” (They all want to believe that) It was probably that. What was the tone of your voice? “You deserved it.” (The most aggravating voice) (You didn’t say it like that, right?) (Just like that) That’s how you said it? That’s just wrong. Maybe like we see in soap operas, perhaps he was in tears and said, “You deserved it.” That’s understandable. Then that would sound like a joke. “You deserved it.” Saying that might have been wrong on my part. But there was a whole process. I went to the hospital because I was worried. You were just making a mean joke, right? You definitely did not mean it, right? – Of course not. / – Sure. He just realized what a terrible mistake he made. You almost failed to be a human being. He’s been going down his own path but after seeing people’s reactions, he turned. (So annoying) When were you angriest at your husband? When I was pregnant with our eldest, my water broke. I called my husband while I was getting an IV. “I’m in the hospital.” “Why?” “My water broke, so I’m getting an IV.” “Okay.” And he just hung up and never came. Even when it was his day off? Yes. When I got home, he was eating with his parents. He never asked me if I was okay. He still hasn’t asked me. – It was during your first pregnancy? / – Yes. I don’t understand. Shouldn’t a father rush to the hospital? Whenever my wife goes to the hospital, she asks for money and I give it to her. Why didn’t you go with her when you were home? She always goes on my day off. Please… Wait. (I’m getting so angry) You should be fired up, flustered, and making excuses. Please don’t make ridiculous excuses in that calm, low voice. I get a lot of stress from work. I have to deal with the customers. Who doesn’t get stressed from work? When we shoot this program, we have to talk to people like you. We’re at work, too. (You’re causing them stress!) We’ve met many people like him. They were into many different hobbies. But they always say, “I don’t drink, smoke or gamble.” So he doesn’t have any other hobbies, right? He likes to drink, too. Once in a while? No. He drank 15 times this month. Does his personality change when he’s drunk? Yes. He has drinking habits? Does he get violent? He becomes violent. I get afraid when he goes out to drink. Why? One time, he went to Bundang to drink. I couldn’t get in touch with him for 4 hours. So I called the cops to locate him. He was asleep in the middle of a highway. – On the road? / – On the road in his car. During my third pregnancy, he drank at his parents’ place. While we were sleeping, he kicked me in the stomach by mistake. – Your stomach? / – Yes. I was 7 or 8 months pregnant. You were close to your due date. I said, “My stomach hurts. Let’s go see a doctor.” He said, “Okay” and fell asleep in the bathroom. I went to the hospital by myself, got an IV and came home. I told him when he was headed to work. “You kicked me in the stomach, so I went to the hospital. Did you know that?” “I don’t remember.” – That’s what he said? / – Yes. But he should’ve apologized for hurting you. “I’m sorry. I don’t remember, but I’m sorry.” – Isn’t that the right thing to do? / – Yes. When I’m eating with Shin Dongyeob and he asks, “Did you finish this?” I apologize even if it wasn’t me. She apologizes very well. We get drunk by food. When we eat, we aren’t sure what we ate so if someone complains, I just apologize. But your husband never apologized? He still hasn’t apologized. He isn’t good at apologizing to others. I don’t quite remember what happened. Even if you don’t remember, she said that you kicked her. She wasn’t lying. Of course you don’t remember since you were drunk. But she told you about it. You realized what you did. So why didn’t you apologize to her? I don’t know why I didn’t. I’m sorry. When she first came out, I thought it must be hard because he’s into fishing so much. But that’s not the only problem. Think about it. We pulled up a stem and endless roots are coming up. We thought fishing was the only problem. We were deceived. Unbelievable. Do you always get serious concerns like this? No, this is too harsh. If we knew there were so many concerns, we wouldn’t have accepted her concern. Their daughters are here. If we talk to them, we can see the truth. Girls. The eldest. (We’re here) How cute. (Eldest daughter) (Second daughter) – They’re so pretty. / – How cute. Who’s the eldest one? The one with black hair. What’s your name? Hello. I’m 8-year-old Jang Hanbyeol. – Hanbyeol. / – Hi, Hanbyeol. – Eunbyeol. / – She’s shy. Our youngest. Setbyeol, how old are you? I’m 5. You’re 5? Does your father go fishing often? Yes. How often does he go? Since I was young. She remembers that he’s been going for years. In your opinion, do your parents get along well? No. They argue often. They argue often? How do they argue? They yell at each other. Who yells first? Dad. What does he yell? “Do it now!” Like that. He told Mom to do something quickly. How do you feel when they argue? – Not good. / – Not good. What do you and your sisters do when your parents fight? – We’re scared. / – You’re scared? – You’re shocked? / – Yes. Of course it’s scary. Hanbyeol got a psychological test at school. They said that she has depression. – Hanbyeol does? / – That little girl? They told me that she needs treatment. It was his day off when I went to get the result. That’s a relief. But he didn’t go with me. He said that he was busy. I told him the result over the phone. They think she has depression and that she needs treatment. – He asked how much it costs. / – Money first? He spends so much money on fishing. The treatment would cost $30 each time. You told him that? It costs $30? He said, “It’s all your fault.” – What? / – That’s what he said? “She became that way because you yell at them. It’s not my fault.” He also said the treatment is too expensive so she can’t get it. That’s not what I meant. We moved recently, so it cost a lot of money. We were in a financial bind. Didn’t you think to go fishing less? I didn’t go fishing then. – Not at all? / – Not at all. I think Hanbyeol had depression because she had just started at a new school and she was ostracized by her classmates. It’s because of what happens at home. She’s intimidated and a loner at school and that’s why. That’s not what I thought. I used to have depression, too. My friends tried to help me out and tried to cheer me up. But if I don’t work hard it doesn’t get better. That’s what you think Hanbyeol needs to do? Teachers think she has depression, but she’s the one who must overcome it herself? (Don’t force an adult method onto a child) We talked to a professional to see what state Hanbyeol is in and if she can really overcome it by herself. We shot a video. Here it is. Hanbyeol, we’ll draw pictures together and play with sand today. It’s psychotherapy counseling. That was very good. We see how she feels through how she plays. Can you draw a house for me? We can see what she thinks through her drawings. (Just a house) She didn’t draw any family members. There are only two flowers. What does it mean? Does anyone live in this house? No. – No one ever lived here? / – No. If I asked you to live here, would you want to live here? No. (Mom is heartbroken) I’ve never seen a child draw a house she dislikes. She’s in a serious state. She has childhood depression. (He’s contemplating) Where is this tree located? – By the beach. / – By the beach. What will happen to it over time? It’ll die. It’ll die. (Oh my…) Is there a way to save it? – No. / – There’s no way? I think it has been going on gradually from unstable attachment. Her home environment has affected her the most. (I’m saddest when Mom and Dad fight) Hanbyeol’s vulnerable psychological aspects could influence her sisters, too. In the future… (A serious result) How do you feel, sir? He must have many thoughts in his head. I’m a bit despaired. – Are you surprised? / – Yes. What part surprised you the most? Her drawing of the house and mentioning us fighting. It saddens me. – You feel bad for her? / – Yes. Do you admit that you’ve fought a lot? Many Koreans tend to diagnose themselves and buy drugs that they think they need. We have the highest rate in the OECD countries. You already diagnosed your daughter like you’re the psychiatrist. “She doesn’t have a problem.” But that’s not true. It’s not that simple. What do you want from your husband? Please go fishing once or twice a month. I’m sure he can’t quit altogether. We’ve never gone on a family trip with the kids in the past 10 years. Except fishing. I’d like to go to a nearby villa and rest for a few days. I want us to be a good mom and a good dad to the kids. Let’s start with Gaga. Is it a concern? This is a serious concern. I feel like I just ate 500 sweet potatoes. I feel so frustrated inside. This is a serious concern. There’s no doubt. It’s a concern. I want to press the button repeatedly, but I don’t want to hurt Hanbyeol’s feelings. Please don’t give her painful memories and embrace her with love. If you think it must’ve been tough for the wife, please press the button. 5, 4, 3, 2, 1. Please stop. Before we see the result, I’d like to say that you lost today. Do you admit that? Yes, I do. Please say a word to your wife. I really love her. She is the best. To your daughters, too. Hanbyeol, Eunbyeol and Setbyeol are my future. I want to live a full life and make a happy family. Are you ready to change? Yes. Are you ready? (Perplexed) He can’t understand English. What did you say? Are you ready? Yes, I’m ready. (We root for your new beginning) How many votes did they get? Please show us. I’m scared to see it. – “An Unfaithful Husband.” / – They got a lot. We have a total of 200 votes. They definitely got more than 100 votes. What’s the result? 160? This is the highest we’ve seen so far.
Fishing. Kashi – baits for catching crucian carp on a float in the spring in March. My fishing Fishing. Kashi – baits for catching crucian carp on a float in the spring in March. My fishing Fishing. Kashi – baits for catching crucian carp on a float in the spring in March. My fishing
Fishing Competetion of bangladesh we are now stayl outside of canal lots of people coming for fishing They think that’s just fishing lets go for fishing, lets go for fishing the canal or beel , like as river of bangladesh hers have lot of big big shol, gozar buyal, & many many others fish they are redy right now for fishing its trap here name polo start fishing Here a hunter fishes catch a huge fish people and people Searching fish this is gojar fish but it is little fish going home
Promise me To grow up and make me fish I want to hunt you and you are the largest size Okay Okay See you later on a better day But you will be for dinner, I apologize for you Hhhhh
– Sounds pretty cool. Wait. – What? – That’s not enough calamari. (laughs) – Where’s the fish? Make sure there’s enough fish in the sea for the next generation. Don’t be shellfish. For a future full of fish,
choose the blue fish tick.
Hello everyone, my name is Cecile Herate and I am a biology researcher in the cytogenetics laboratory of the French Alternative Energies and Atomic Energy Commission. In this lab, we are experts in biodosimetry and we develop different cytogenetic methods to retrospectively estimate the received dose by individuals. We are also using these techniques to answer mechanistic questions by analysing chromosome rearrangements In this tutorial, I will particularly explain you the Multi-FISH (or M-FISH) staining method that allows building the karyotype by differentiating each of the 23 or 24 chromosomes. I will tell you more very soon. After the chromosome preparation by the different methods and the spreading on slides, DNA has to be stained for further acquisition using a fluorescent microscope in order to allow for the different chromosome observations. The m-FISH staining consists in labelling DNA using probes specifically recognising each chromosome sequence. Therefore, it allows for the visualisation of interchromosomic rearrangements such as translocations and unstable aberrations like dicentric chromosomes. In this MOOC, I will describe in more detail the m-FISH protocol using the commercial probes from Metasystems. This staining can be performed on chromosomes obtained after the classic protocol of metaphasis for “DCA assay”, but also on premature chromosomes obtained with the “PCC assay”. Here is an example. On the left picture, you can see the m-FISH staining of metaphasic chromosomes and on the right the resulting karyotype. You can observe two dicentric chromosomes and one translocation. The latter would not be detectable with a conventional staining. Different cytogenetic methods are used in biodosimetry and allow the determination of the received dose by individuals. The « Dicentric Chromosome assay » or « DCA » allows, with the use of a Giemsa staining, the scoring of dicentric chromosomes and rings. While the telomere and centromere staining does not modify the main principle of the technique but facilitates the analysis and improves the detection of chromosome aberrations. The « Premature Chromosome Condensation assay » or « PCC » allows for scoring of chromosome fragments and the determination of excess fragments. In this case, the FISH staining results in specific scoring of dicentric chromosomes, rings and acentric fragments. So, the analysis is a little bit longer but more precise and informative. The « Cytokinesis Block Micronuleus assay » or « CBMN » allows for scoring of micronuclei after blocking the cell cycle in the cytokinesis step. The 3 techniques are explained in detail in 3 other specific tutorials that are also available in this Youtube channel. In this slide, you can see the three types of staining: Giemsa, Telomere and Centromere and m-FISH for both DCA and PCC assay. Try to spot the chromosome aberrations… Good luck. Now, we will see the precise protocol of the m-FISH staining using Metasystems probes. I will try to provide you with the maximum of details and the tricks that we have developed. First, you can see the list of materials you need for the staining, the necessary laboratory equipment and the various reagents. Pay attention to work under the chemical hood when indicated, since some solutions are toxic. Here is the list and the composition of the different solutions that you need to prepare. The day before the staining, you can already thaw the slides or take them out from the refrigerator. I remind you that cytogenetic slides can be kept for years at -20°C. Usually, the slides that are stained using m-FISH have previously been observed with other staining methods and therefore will need to be destained through washing. After coverslip removal by a regular PBS prolonged incubation, we incubate 30 minutes in PBS/Tween 0.5% at 37°C. Before starting, you can prepare the solutions as indicated in this slide. The first step consists of preparing the slides by fixing the chromosomes in Formaldehyde. This step needs to be performed even if the chromosomes had already been fixed previously for performing another staining. Don’t forget to work under the chemical hood! We are using porcelain boxes for the different incubations. After washing, the slides have been rinsed in PBS. Now, I am showing you the step of fixation. I transfer the slides from PBS in Formaldehyde 4%. I will incubate them for exactly 2 minutes. After rinsing in PBS, the slides are transferred in 0.1 SSC at room temperature. The next step is the DNA denaturation. We first transfer the slides from 0.1 SSC to 2X SSC solution preheated at 70°C in a water bath. We incubate for 30 minutes. Then, we move the 2X SSC box to a 37°C water bath and let it cool down for another 30 minutes in order to allow for the renaturation of repeated sequences. We rinse for 1 minute in 0.1 SSC and continue the denaturation. So, we incubate for one minute in NaOH 0.07N. Then, we continue with 2 successive rinses, first in 0.1 SSC and then in 2X SSC, both at 4° C. Finally, we proceed to drying and incubating in ethanol baths of increasing concentrations. Ethanol has the property of triggering dehydration. Here we transfer successively the slides from Ethanol 30% to Ethanol 50% then 70% and finally 100%. Each incubation lasts one minute. Finally, we remove the slides and place them in a specific rack. The second thing to do is to prepare the m-FISH probes. You have to calculate 15µL per slide. The denaturation consists in heating the probe solution for 5 min at 75°C. After 5 minutes we stop the denaturation by placing the tube in ice for a few seconds. Then, we transfer in the 37 °C water bath for 30 min to provoke the pre-hybridization. Now that both the slides and the probes are ready, we can start the hybridization process. Before starting, try to engrave on the back of the slides the chromosome spreading area in order to optimise as much as possible the deposit of the probe solution. Lay your slide down and take 15uL of the probe solution. Distribute in tiny drops on the slide as I am showing you. Then, protect with a plastic coverslip that you have previously sized from a freezer film for the delimited area. Try to be meticulous when you drop the coverslip to avoid bubbles. If you get some, please try to remove them. The hybridization will then occur in a box containing 2X SSC with 50% formamide as indicated previously in the protocol. Here, we use a “homemade” hybridization box. It consists in a plastic box with a central platform. On the side gutter, we have placed paper strings that we will generously humidify with the hybridization solution. Since we will incubate the slides for a long time, it is necessary to tape the box or find any other way to seal it in order to prevent the hybridization solution from drying out. For this staining, the incubation is quite long and will take place in a 37°C incubator. A bacteria incubator set at 37°C could also do the job. You just need to find a way to maintain the temperature. We will incubate the samples for 4 to 5 days in the dark. 4 days is enough but if you want to be sure to have a strong signal you can wait 5 days. 4 or 5 days later, we will proceed to the slide washing. Refer to the solution list for the composition of the two washing solutions. We unseal the hybridization box and since it contains formamide we do it under the chemical hood. We remove each coverslip using a pair of tweezers and wash first in 0.4XSSC for 3 min at 72°C. Then, we perform a second washing in 2XSSC containing Tween at 0.05% for 3 min at room temperature. After that, we rinse in PBS. The DAPI staining that follows consists in simply incubating the samples for 5 min in the DAPI solution prepared at a concentration of 100 mg/mL before washing in PBS for another 5 min. The DAPI solution can be kept up to one week at 4°C. When the slides are ready for mounting, we take them out and dry the back. We take P-Phenylenediamine or PPD with a Pasteur pipet. We drop PPD on the slide and put the coverslip from the bottom to the top. In order to remove the excess of PPD and destroy entrapped bubbles, we put the slide between paper towel and press gently. The slides are now ready for observation and acquisition with a fluorescent microscope. After the DAPI staining, the slides are ready to be analysed. In the meantime, they can be stored either at 4°C for a few weeks or at -20°C for years in specific plastic storage boxes. For the microscope acquisition, you need to have access to a fluorescent microscope with DAPI channel and the 5 following filters: aqua, green, orange, red and near infrared. In our lab, we use the METAFER acquisition software and I will show you the main steps for image acquisition using this software. First, if you didn’t do it previously for the analysis of other staining, you have to create a gallery with the 10X objective, the software will automatically capture the cells of interest. You can make either an automatic research of metaphasis or PCC. After cleaning the slide with Ethanol, you can start with either the establishment of the gallery or the M-FISH staining acquisition. The gallery is established with the 10X magnification. We program the microscope to acquire metaphasis and then it captures all of them in the predefined area. After the triage of the gallery, we start the final acquisition with the 6 fluorescent channels. The microscope will acquire successively the different channels to obtain the final image that allows for the definition of the karyotype of the cell. The M-FISH staining seems to be very informative and finally it is the only staining that allows detection of stable aberrations such as translocations. But, it also takes so much time… When are you really using it, because, I guess that, you cannot apply it in case of emergency for direct dose assessment. You are right, this assay is mainly used for retrospective dose assessment when dicentrics chromosome detection is no more accurate. Indeed, the rate of dicentric, rings and acentric fragements decrease along each cell division. About 50% of dicentric chromsomes are transmitted to the daughter cells. It means that after some months the dose determined with classical technique is inferior to the initial dose. Even if some algorithms allow the modelisation of the decrease. The better method is to quantify stable aberrations that remain stable during decades. Moreover, this technique is also interesting for fundamental research to characterize the phenomenon of DNA reparation and recombination. You say that I can apply the M-FISH staining on my old slides. Is there really any problem? Yes, slides can be keep at -20°C for years without any damage. Just thaw them the day before the staining. In case of previous staining you just have to remove it, however when you are making a Giemsa staining it is better to remove it rapidly in the following days. Another thing to note. Multi-FISH staining is an expensive assay but also very informative, it is usually the last staining performed on slide. After this staining, following ones are no more as reliable. Just a short come back to the protocol, it seems like a simple recipe, different incubations for fixation, protein digestion, denaturation and hybridization. But, what seems more obscure to me is the images acquisition and the analysis. Is it actually so evident? You are right. It is not a complicated assay, you just need to follow step by step the protocol, and be vigilant to respect the time and temperature of each incubation. Regarding the microscope capture, the way to do differs from the different instrument and software. That’s why I didn’t describe deeply this step. Anyway, the principle remains the same. If you didn’t make it before you need to make a gallery using the 10X magnification and the channel for the DAPI acquisition. Either you have in your software an automatic capture of metaphasis and PCC or you can make it by yourself in saving the coordinates of each cell of interest. In case of automatic detection, you need to make the triage of the gallery. At that point, you are ready for the finale capture using the 5 filters already described with the 63X objective. I cannot tell you more about time of integration, it really depends on each microscope. After export of the images, you can analyse them with a dedicated software. We are using Isis but for sure there are some others that will create automatically the karyotype.
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These tips works for any pan fish such as this perch 5 minutes for preparation and 10 minutes for cooking 1. Dry the fish with paper towel 2. Cover it with dry seasoning such as salt and pepper 3. Wipe the pan with ginger 4. Add fish to the hot pan with warm oil Cooking time for this size is around 10 minutes 5. Don’t move the fish much until you are
ready to turn to the other side 6. Use a non-sticky pan, which allows you to use less oil With some cumin, this dish becomes ready!
I’m Gerard van der Schaar of the Clavis Private
Energy Solar Boat Team Since 2008 we’re sailing at the Solar Challenge
and with the experience we have developed our team.
Everyone has got its own discipline and know what he has to do during the race and how to get on the podium. Mark Scholten is responsible for the software
of the battery and also for the telemetry system during the preparation. During the race he’s responsible for the strategy. Danny is responsible for Twitter and Facebook.
And make sure we can see what the stage of charge on our watch is and how much energy we receive. Evelien is responsible for the PR materials
she makes sure that after the race the media get our result. Kasper is responsible for the telemetry and
everything will be visualised on our website. So we can take the right steps. Frank is responsible for the maximum power
point tracker. So all the energy from the solar panels will be transmitted to battery
where it can be used Each year there fall off some new teams and
appear new teams. You know see there are more international teams. And also good teams from
Finland and America who came third last year. And TU Delft comes with a daring concept.
so we expecting higher speeds this year. the biggest challenge for us is that everything
will be in order. And race a good and safe. because the biggest challenge is to let the
boat perform optimal every day. We have put a lot of energy in testing and the reliability
the last two years. know we have to prove our work is don right during the race. We expect a competitive race from Groningen
to Friesland. Our goal is to be on the podium and show our progress of the last years. When you sail into the Prinsentuin in Leeuwarden
over 40km/h on hydrofoils, That’s how you prove you have done it right. A little boy
will say: that’s how it should be done. That’s what we whant to achieve. Not by telling but showing it can be done.