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....