Hi, I am working on chicken duodenum and I faced with 2 problems during working on IHC.
1- I faced with high background and for eliminating that, based on articles, I used 0,1% Sudan Black B in 70% ethanol, for 20 min and follow my protocol for staining. But, I could not see any deceasing of background.
2- I am working with NDV virus and I used both conjugated and un-conjugated Ab. But. I can not see any virus in duodenum. My conjugated Ab is ALEXA FLUOR 555 which suggests 1:50 and 1:200 dilatation. But, I could not detect any virus in that dilutions. Some references suggested 1:8000 and after I tried, even that didn't work for me. Now, I do not know where is my problem???!!!
My un-conjugated Ab is Lightning-Link R-Phycoerythrin (LL-RPE) kit which I used 3×100 µg kit. I used dilutions from 1:50 to 1:2000 and did not succeed.
Here I mention my protocol in case any part of this wrong. Besides, I attached a file which I indicated that how I prepared my reagents for IHC, in case any part of my preparation also be wrong. I be happy if anybody can help me.
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1. Heat slides in an oven at 60 for 30-60 minutes.
2. Deparaffinization:
- Xylene 5 minutes
- Xylene 5 minutes
- Xylene 5 minutes
3. Rehydration:
- 100% Alcohol 3 minutes
- 100% Alcohol 3 minutes
- 95% Alcohol 2 minutes
- 95% Alcohol 2 minutes
- 80% alcohol 2 minutes
- dH2O 5 minutes
- dH2O 5 minutes
4. Antigen Retrieval:
- Boil slides in water bath with a mix of 0.1 M sodium citrate buffer and 0.1 M citric acid solution, pH 6.0 at 90°C for 20 minutes.
- Remove the slides from heat and allow them to stand at room temperature in buffer for 10 minutes.
- dH2O 5 minutes
- TBST 3 changes , 5 minutes
5. Immunostaining:
- Block endogenous peroxidase activity with 3% hydrogen peroxide (H2O2) fo 10 minutes at RT
- TBST 3 changes , 5 minutes
- Block sections with 5% BSA blocking solution for 2 hours at RT.
- TBST 3 changes , 5 minutes
- Drain the slides and incubate the diluted primary antibody overnight at 4 oc
- TBST 3 changes , 5 minutes
- Immerse the slides in a jar of 0,1% Sudan Black B in 70% ethanol, for 20 min, in room temperature
- TBST 3 changes , 5 minutes
- Counterstain with hematoxylin for 1 minute
- ddH2O 5 minutes
- ddH2O 5 minutes
6. Dehydration:
- 95% Alcohol 1 minute
- 95% Alcohol 1 minute
- 100% Alcohol 1 minute
- 100% Alcohol 1 minute
- Xylene 1 minute
- Xylene 2 minute
- Xylene 2 minute
- mount coverslips
Hi Mostafa,
first, do you know in which cell type the virus should be found?
Second, what type of background do you see? Stained mucus? Stained macrophages?
Third, if you use a direct immunofluorescence system (directly conjugated primary antibody) the amplification may be too low for visualizing low-abundance antigens.
To see, whether virus is present at all, I would suggest using an ABC (Avidin-Biotin-Complex) technique for peroxidase such as Vectastain from Vector Labs with diaminobenzidine as chromogen and a normal transmitted light microscope. Endogenous peroxidase and endogenous biotin may be a problem in the gut, but not so much in paraffin sections. Besides, there are ways to circumvent such problems.
I can send you references, if you are interested.
Best regards
Birte
Hi, thank you for your reply. Actually, after I used Sudan Black B as I mention in earlier, the background already solved. But, the auto Fluorescent problem still exist. I check with other tissues as well like spleen and lung and I noticed that auto Fluorescent in duodenum, spleen and lung come from RBCs. based on morphology, macrophages have seen rare and mostly seen RBCs and I do not know how shall i eliminate the RBCs in duodenum.
I appreciate if you can reference to see the present the virus in tissue. becasue even i used this protocol for different highly infected chicken tissues such as duodenum, lung and spleen and still havent seen ant virus in tissue level.
best regards,
Mostafa
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