Immuno staining on Paraffin section

Here I am sending a procedure that I used to performed Immuno staining....Hopefully, it help you...

Indirect immuno-incubation of paraffin sections

1. Cut 5-7 µm sections of paraffin embedded tissue and put them on Menzel Superfrost

slides.

2. Dry overnight at 37°C .

3. Deparaffinization; xylene 2x2 min

alcohol 100% 2x1 min

alcohol 90% 2x1 min

alcohol 80% 1 min

alcohol 70% 1 min

alcohol 50% 1 min

aqua dest 1 min

4. Microwave treatment in 0.01M sodiumcitrate (6 ml NaCi 1M in 600 ml aqua dest);

7 min. at 850 Watt and 2 times 3 min. at 850 Watt. (maximum of 12 slides.)

5. Cool down in citrate solution for at least 30 min.

6. Rinse in PBS- (PBS 1x) for 2 min.

7. Block for endogenous peroxidase during 30 min. at RT. in PBS/H₂O₂/azide

100 ml PBS 1x (0.1M PBS)

2 ml H₂O₂ 30%

1 ml sodiumazide 12.5% (toxic!!)

8. Rinse in PBS- for 2 min.

9. Rinse in PBS+ (PBS 1x/ 0.5% protifar/ 0.15% glycine), 2 times 2 min.

1L PBS, 5g protifar and 1,5g glycine.

10. Incubation with the primary antibody for 90 min. at RT, (or overnight at 4C). (100µl/slide)

11. Rinse in PBS+ 3 times 5 min.

12. Incubation with the peroxidase conjugated secondary antibody for 60 min. at RT.

13. Rinse in PBS+ 3 times 5 min.

14. Rinse in PBS- for 2 min.

15. Incubation with DAB-substrate (DAKO Liquid DAB substrate-chromogen system)

Use 20µl DAB solution in 1 ml Dako buffer, 100µl/slide during 4-8 min.

! Wear gloves; DAB is carcinogenic!

16. Remove DAB-solution by placing the slides in aqua dest.

17. Refresh aqua dest immediately.

18. Counterstain with haematoxylin for 5 min.

19. Rinse in tapwater for 10 min.

20. Dehydration; alcohol 96% 1 min.

alcohol 100% 2x1min.

xylene 2x2min.

21. Mount with Entellan.

22. Dry overnight at 37°C.

! Note that all dilutions are made in PBS +

Introduction of Immuno staining

Done...OK as promised I will tell you about Immuno staining or immunohistochemistry. What is definition of Immunohistochemistry ?. Immunohistochemistry is the localization of antigens or proteins in tissue sections by the use of labeled antibodies as specific reagents through antigen-antibody interactions that are visualized by a marker such as fluorescent dye, enzyme, or colloidal gold.

Tissue preparation is the cornerstone of immunohistochemistry.To ensure the preservation of tissue architecture and cell morphology, prompt and adequate fixation is essential. We usually use 4% paraformaldehyde in 0.1M phosphate buffer as fixative.

Paraffin sections than needed and it has been the largest proportion of material for immunohistochemistry formed as formalin-fixed, paraffin-embedded.It produces satisfactory results for the demonstration of majority of tissue antigens with the use of antigen retrieval techniques.

Small blocks of tissue (less than 5 mm thick) can than be processed as whole mounts.

The demonstration of many antigens can be significantly improved by the pretreatment with the antigen retrieval reagent that break the protein cross-links formed by formalin fixation and thereby uncover hidden antigenic sites.

Background staining may be specific or non-specific. Inadequate or delayed fixation may give rise to false positive results due to the passive uptake of serum protein and diffusion of the antigen. Such false positives are common in the center of large tissue blocks or throughout tissues in which fixation was delayed.

Antibodies, specially polycolonal antibodies, are sometimes contaminated with other antibodies due to impure antigen used to immunize the host animal.

The main cause of non-specific background staining is non-immunological binding of the specific immune sera by hydrophobic and electrostatic forces to certain sites within tissue sections. This form of background staining is usually uniform and can be reduced by blocking those sites with normal serum.

Special controls must be run in order to test the protocol and for the specificity of the antibody being used.

we have 2 methods to perform it. Direct method is one step staining method, and involves a labeled antibody (i.e. FITC conjugated antiserum) reacting directly with the antigen in tissue sections. This technique utilizes only one antibody and the procedure is short and quick. However, it is insensitive due to little signal amplification and rarely used since the introduction of indirect method. Indirect method involves an unlabeled primary antibody (first layer) which react with tissue antigen, and a labeled secondary antibody (second layer) react with primary antibody (Note: The secondary antibody must be against the IgG of the animal species in which the primary antibody has been raised). This method is more sensitive due to signal amplification through several secondary antibody reactions with different antigenic sites on the primary antibody. In addition, it is also economy since one labeled second layer antibody can be used with many first layer antibodies (raised from the same animal species) to different antigens.
The second layer antibody can be labeled with a fluorescent dye such as FITC, rhodamine or Texas red, and this is called indirect immunofluorescence method. The second layer antibody may be labeled with an enzyme such as peroxidase, alkaline phosphatase or glucose oxidase, and this is called indirect immunoenzyme method.

to be continued........

Immuno staining

I would like to tell you about immuno staining, but OK, I will post it later. I will prepare it....

Hari ......

Hari ini agak 'bete' apa ya.....
habis liburan 4 hari di long weekend membuat 'agak males' untuk berangkat ke lab....Pagi hari terlihat di luar udara cerah, sunny, dan angin tidak berhembus kencang...tidak sperti kemaren yang kecepatannya sampai 40 km/jam...
Tadi malem hujan salju....aneh..padahal kalo di hitung bulan, harusnya sudah mendekati spring....tapi saya menjadi maklum...apalagi wa selalu ingat pepatah my landlord....Jangan pernah percaya dua hal di belanda ini....opo kuwi bang...?? begitu awal kali belum tahu...."wanita dan cuacanya".....ngakak kalo inget pertama kali.....

Yah tapi wa paksaan berangkat....sampe lab suasana kayaknya masih sepi walaupun jam sudah menunjuk 09.30...kayaknya mereka juga merasakan hal yang sama....beberapa selang kemudian mereka satu -persatu muncul....dan aktifitas pun dimulai....

Hari ini gak ada hal yang wa kerjakan karena emang gak ada jadwal....jadi ya ber chit chat dengan teman di lab.....

Hari berakhir...dengan makan malam dengan menu makaroni ala Itali.....enak.....thanks bang......

Diamond, just refresh my mind.......

Actually I don't like to share something that it is only for fun. But OK, I don't know why I would like to tell you about it. For a couple days, I liked to see diamonds and jewelery in the internet. There is something in my mind which always push me to see them. I know this may be funny for you. But I got the point in this activity, that there is no things big except something who create it happened. He is God. You can find a lot of information about diamond in the internet as www.diamond.com. You can also try www.liontin.net as one stop resource about diamond and jewelery. Most of them provide certified diamond. Enjoy it...

You can isolate DNA by yourself

He..he...

Today I would like to give you 'teaching' how to isolate DNA in your
own home by yourself with your any kitchen tools in your home. Yesterday I read some resource that it is worth to have a reading. OK lets make your first experiment how to isolate DNA by yourself easily...

1. First, you need to find something that contains DNA. Since DNA is the blueprint for life, everything living contains DNA. For this experiment, we like to use green split peas. But there are lots of other DNA sources too, such as: Spinach, chicken liver, meal, chicken lungs, or what ever....

2. You can not use your stone, gold, silver, mug, to do it as it is not live things...

3. 3. Put in a blender : 1/2 cup of split peas (100ml) , 1/8 teaspoon table salt (less than 1ml), 1 cup cold water (200ml). Blend on high for 15 seconds. The blender separates the pea cells from each other, so you now have a really thin pea-cell soup.

4. Pour your thin pea-cell soup through a strainer into another container (like a measuring cup).
Add 2 tablespoons liquid detergent (about 30ml) and swirl to mix. Let the mixture sit for 5-10 minutes. Pour the mixture into test tubes or other small glass containers, each about 1/3 full.

5. Add a pinch of enzymes to each test tube and stir gently. Be careful! If you stir too hard, you'll break up the DNA, making it harder to see. Use meat tenderizer for enzymes. If you can't find tenderizer, try using pineapple juice or contact lens cleaning solution...
* In this experiment, meat tenderizer acts as an enzyme to cut proteins just like a pair of scissors. The DNA in the nucleus of the cell is molded, folded, and protected by proteins. The meat tenderizer cuts the proteins away from the DNA.

6.Tilt your test tube and slowly pour rubbing alcohol (70-95% isopropyl or ethyl alcohol) into the tube down the side so that it forms a layer on top of the pea mixture. Pour until you have about the same amount of alcohol in the tube as pea mixture.
DNA will rise into the alcohol layer from the pea layer. You can use a wooden stick or other hook to draw the DNA into the alcohol.

7. What is that stringy stuff ??. Alcohol is less dense than water, so it floats on top. Since two separate layers are formed, all of the grease and the protein that we broke up in the first two steps and the DNA have to decide: "Hmmm...which layer should I go to?"

This is sort of like looking around the room for the most comfortable seat. Some will choose the couch, others might choose the rocking chair.

In this case, the protein and grease parts find the bottom, watery layer the most comfortable place, while the DNA prefers the top, alcohol layer.

DNA is a long, stringy molecule that likes to clump together.

8. Now that you've successfully extracted DNA from one source, you're ready to experiment further. Try these ideas or some of your own:

Experiment with other DNA sources. Which source gives you the most DNA? How can you compare them?

Experiment with different soaps and detergents. Do powdered soaps work as well as liquid detergents? How about shampoo or body scrub?

Experiment with leaving out or changing steps. We've told you that you need each step, but is this true? Find out for yourself. Try leaving out a step or changing how much of each ingredient you use.

Do only living organisms contain DNA? Try extracting DNA from things that you think might not have DNA.

Hooray...you get the DNA and you already become scientist...Congratulation.....!!!

Laptop for Sell

Hmmm...I got new problem with my laptop. My laptop so lazy so I am preparing to upgrade it. I searching in the internet to look for some hardware property, and I found some sites as a good site. On of them is www.laptopforus.com. I got many links as resource to find laptop properties. Some of sites gave provide discount card. It help me so much...thanks..

Just want to share.....may you want to have a look..

Fragile X Syndrome

Today...

I am reading new post on fraxa foundation that effective treatments and a cure for all children and adults with Fragile X will be 'in front our eyes', by directly funding the most promising research.

Researchers are optimistic that the mGluR Theory of Fragile X will lead to treatments for Fragile X and for autism.
What is about mGluR theory ? OK lets me give you this references :

Development of the mGluR Theory :

• May 1997 FRAXA investigator and scientific advisor Dr. William Greenough reports that FMRP, the fragile X protein, is synthesized in dendrites in response to synaptic activity and stimulation of metabotropic glutamate receptors (mGluRs).
  
  
• Nov. 2000 In a FRAXA-funded research project started at Brown University, Drs. Mark Bear and Kim Huber make the important discovery that one mechanism of communication between neurons is defective in mice which have been bred to model Fragile X. This mechanism, called long-term depression (LTD), is a form of synaptic plasticity, the molecular basis of learning and memory. The team studied one specific form of LTD which occurs only if and when mGluRs are stimulated. They found that mGluR-LTD is excessive in the Fragile X knockout mouse.
  
  This discovery has enormous implications for our understanding of Fragile X and related autism spectrum disorders. It and follow-up experiments have led to the "mGluR Theory" of Fragile X: that exaggerated signaling in mGluR pathways underlies many cognitive, behavioral, and neurological symptoms of Fragile X (and probably autism, too.)
  
  
• May 2001 With FRAXA funding, of Columbia University tests the mGluR Theory by treating Fragile X mice with MPEP, a compound which blocks one kind of metabotropic glutamate receptor (mGluR5). According to the theory, this should reverse the major symptoms of Fragile X. In mice, the simplest symptoms to test are hyperactivity and sound-induced seizures. MPEP is found to reverse these symptoms with a single low dose in Fragile X mice.
  
  
• April 2002 The mGluR Theory is introduced at FRAXA's Banbury Conference on Fragile X at Cold Spring Harbor Laboratory, spurring other researchers to follow up on this discovery. Fragile X is now becoming accepted as the first known disease of "synaptic plasticity." Yearly Banbury meetings have since recruited some of the world's top neuroscientists to the study of Fragile X.
  
  
• 2003 A team led by Dr. Tom Jongens of the University of Pennsylvania demonstrate that fruit flies with a mutated Fragile X gene have learning deficits and that MPEP can rescue these abnormalities, even when given to adult flies. The team subsequently shows that the Fragile X flies have abnormal brain anatomy, which can also be corrected by treatment with MPEP during development. Further studies demonstrate that an available drug, lithium, which inhibits mGluR signaling pathways, also rescues Fragile X fly anatomy and cognition. FRAXA then commissions further studies at the Bauchwitz lab at Columbia, which confirm that lithium can treat seizures and hyperactivity in the Fragile X mouse model.
  
  
• 2004 FRAXA-funded researcher Dr. Robert Wong at SUNY Downstate demonstrates that isolated slices of Fragile X knockout mouse brain have more seizure activity than normal mouse brain. He shows that this seizure activity occurs only if mGluR5s are stimulated and shows also that MPEP blocks it.
  
  
• February 2005 Dr. Peter Vanderklish of Scripps Institute, a FRAXA-funded investigator, shows a distinct pattern of abnormal protein synthesis in Fragile X neurons. This pattern immediately normalizes with brief MPEP treatment.
  
  
• July 2005 FRAXA funds a clinical trial of lithium in Fragile X patients, run by Dr. Elizabeth Berry-Kravis at RUSH University, Chicago.
  
  
• July 2005 Researchers at Hoffman LaRoche report that fenobam, a compound used in Phase II/III human trials from 1978-82, is a selective mGluR5 antagonist. In these trials, fenobam showed efficacy for treatment of anxiety disorders, but it was never tested in patients with Fragile X. Its patent has now expired, so it can be tested at will.
  
  
• December 2005 FRAXA contracts with Scynexis to synthesize fenobam for experimental basic research and makes test batches available to qualified researchers free of charge.
  
  
• January 2006 FRAXA is now actively collaborating with several of the largest pharmaceutical companies in the world and several of the smallest startup companies to bring treatments for Fragile X into clinical trials. FRAXA is developing clinical trial sites, as well as improved biomarkers and outcome measures, which will make future trials more effective. FRAXA is also funding the testing of available medications like lithium, which may have been overlooked in the past as potential treatments for Fragile X. We will continue to develop the capacity necessary to advance potential therapies through clinical trials and into routine use.

Taken from Fraxa.org.

I also got newsletter form fraxa that phase II human trial of Fenobam is underway.
Here is the newsletter :

"In December we announced that an initial Phase 1 trial of fenobam in normal volunteers had started. That trial has been completed successfully, and a Phase II trial of fenobam in patients with Fragile X has begun.

Neuropharm and FRAXA Research Foundation are working together to explore the potential of the mGluR5 antagonist fenobam to treat Fragile X Syndrome. Neuropharm, a speciality pharmaceutical company focused on neurodevelopmental disorders, received Orphan Drug Designation in November 2006 for fenobam to treat Fragile X, after acquiring rights to relevant data on the compound from FRAXA. The Orphan Drug Act aims to speed up development of treatments for rare diseases, defined as affecting 200,000 or fewer U.S. residents, like Fragile X.

Fragile X Syndrome is the most common inherited cause of mental impairment and autism. Its symptoms include intellectual handicap, hyperactivity, attention problems, autistic features, emotional liability and epilepsy. There is currently no effective treatment for the condition.

This Phase II trial is being conducted in the US by Professor Randi Hagerman from the UC Davis MIND Institute (www.ucdmc.ucdavis.edu/mindinsti tute/) and Professor Elizabeth Berry-Kravis from Rush University Medical Center ( www.rush.edu/). Since mGluR5 antagonists have not been given to people with Fragile X before, this first study will use a single dose of fenobam in each patient. The outcome of this investigation will determine the feasibility of longer-term studies with fenobam.

Robert Mansfield, Neuropharm's CEO, commented: "We are delighted to announce the commencement of this study with Professors Hagerman and Berry-Kravis who are world leading experts in the clinical study of Fragile X Syndrome. Their trial of NPL-2009 (fenobam), a targeted treatment for Fragile X Syndrome, is a milestone for those affected by this condition. This study follows the work of FRAXA, and researchers such as Dr. Mark Bear, which has highlighted the potential of mGluR5 receptor antagonists for this patient group."

Lets pray together, may it will be success soon..

DNA and RNA Definition

Wiuh....today I got the message from my friends asking me how to calculate and analyze his research. OK I will try this "new job". BTW today I want to let you know about DNA. What is DNA exactly ?
DNA is Deoxyribonucleic acid. One of two types of molecules that encode genetic information. DNA is a double-stranded molecule held together by weak hydrogen bonds between base pairs of nucleotides. The molecule forms a double helix in which two strands of DNA spiral about one other. The double helix looks something like an immensely long ladder twisted into a helix, or coil. The sides of the "ladder" are formed by a backbone of sugar and phospate molecules, and the "rungs" consist of nucleotide bases joined weakly in the middle by the hydrogen bonds.

There are four nucleotides in DNA. Each nucleotide contains a base: adenin (A), guanin (G), cytosine (C), or thymine (T). Base pairs form naturally only between A and T and between G and C so the base sequence of each single strand of DNA can be simply deduced from that of its partner strand.

The in genetic code DNA is in triplets such as ATG. The base sequence of that triplet in the partner strand is therefore TAC.

The first proof that DNA was the hereditary material was provided in 1944 by Oswald Avery, Maclyn McCarty and Colin MacLoed. The double helical structure of DNA was discovered in 1953 by James D. Watson and Francis H.C. Crick with the invaluable collaboration of the X-ray crystallographer Rosalind Franklin. Watson and Crick shared the 1962 Nobel Prize in Physiology or Medicine with Maurice H.F. Wilkins.

And what about RNA ?

RNA: Short for ribonucleic acid, a nucleic acid molecule similar to DNAbut containing ribose rather than deoxyribose. RNA is formed upon a DNA template. There are several classes of RNA molecules.

They play crucial roles in protein synthesis and other cell activities:

• Messenger RNA (mRNA) is a type of RNA that reflects the exact nucleoside sequence of the genetically active DNA. mRNA carries the "message" of the DNA to the cytoplasma of cells where protein is made in amino acid sequences specified by the mRNA.

• Transfer RNA (tRNA) is a short-chain type of RNA present in cells. There are 20 varieties of tRNA. Each variety combines with a specific amino acid and carries it along (transfers it), leading to the formation of protein with a specific amino acid arrangement dictated by DNA.

• Ribosomal RNA (rRNA) is a component of ribosome. Ribosomal RNA functions as a nonspecific site for making polypeptides.

You see that DNA contains materials which develop our body, our characteristic, our mind, everything in our body including mind and thinking. And the miracle is that it can and always be heredited by ourself to our children.

OK...may it help you..

DNA Isolation from hairs

Today...

I am planing to walk to the laboratory. Whuih....The weather so cold outside. It make me so lazy to walk, I remembered who nice weather in Indonesia... I prepare all the tools and foods. Here we go...!!!!

Today I am trying to isloate DNA from the hairs. It have never been done before, by my self or anyone in my laboratory. So its nice to do it...OK so what i have to prepare...
I prepare all the solutions below :
320 ul mix solutions :
- 50 uM Tris PH 7,5
- 10 uM EDTA
- 150 uM NaCl
- 1 % SDS
- 10 ul Proteinase K

And this how to perform Proteinase K solutions. Because when we order from the manufacturer. It is on powder form.
Proteinase K 10 ug/ul.
- 0,5 gr prot K (powder)
- 2,5 ul 20 % SDS
- add H2O total 50 ul and then incubate in 37 C 30 minutes.

OK let's we go how to perform it.
1. cut hair roots 0,5 cm 10-20 from hairs. Put on the mix solutions, incubate it in 55-56 C over night. (what for ?) it will be nice temp for prot K working.
2. take the mix tube from incubator.
3. let them in room temperature for 30-60 minutes.
4. add 100 ul 6 M NaCl.
5. spin down 15 minutes on high speed (13-14000 rpm)
6. take supernatant and move to new tube. You don't need pellet at this step, so trow away your pelet.
7. add 850 ul 96 % Ethanol or you can use absolute Ethanol to supernatant, shake smoothly and then spin down 1 minute on max speed.
8. you will get the pellet on this step and keep this pellet as your DNA remains in the pellet. So trow away your supernatant.
9. please keep remember position the tube when you do ultracentrifugation as pellet sometimes did not appear clearly.
10. add the pellet with 70% ethanol 350 ul and again spin down for 1 minutes on max speed.
11. hooray you get the pellet (sometime not clear)..trow away your supernatant and let them dry in RT for 15 minutes....
12 add 100 ul water or buffer and your DNA is ready for PCR...

OK enjoy it...

Hello World

Hello all...

Sometime we confuse, sometime we need to share what have been done or we thought. Herewith, I send what I have done and what I will do. Basically, I am interesting in molecular medicine and all about genetic disease. I would like to let you know about molecular and laboratories techniques .
Let me show you what I know...
Thanks for reading..