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..